High Glucose Impairs Expression and Activation of MerTK in ARPE-19 Cells.

MerTK (Mer Tyrosine Kinase) is a cell surface receptor that regulates phagocytosis of photoreceptor outer segments (POS) in retinal pigment epithelial (RPE) cells. POS phagocytosis is impaired in several pathologies, including diabetes. In this study, we investigate whether hyperglycemic conditions may affect MerTK expression and activation in ARPE-19 cells, a retinal pigment epithelial cellular model. ARPE-19 cells were cultured in standard (CTR) or high-glucose (HG) medium for 24 h. Then, we analyzed: mRNA levels and protein expression of MerTK and ADAM9, a protease that cleaves the extracellular region of MerTK; the amount of cleaved Mer (sMer); and the ability of GAS6, a MerTK ligand, to induce MerTK phosphorylation. Since HG reduces miR-126 levels, and ADAM9 is a target of miR-126, ARPE-19 cells were transfected with miR-126 inhibitor or mimic; then, we evaluated ADAM9 expression, sMer, and POS phagocytosis. We found that HG reduced expression and activation of MerTK. Contextually, HG increased expression of ADAM9 and the amount of sMer. Overexpression of miR-126 reduced levels of sMer and improved phagocytosis in ARPE-19 cells cultured with HG. In this study, we demonstrate that HG compromises MerTK expression and activation in ARPE-19 cells. Our results suggest that HG up-regulates ADAM9 expression, leading to increased shedding of MerTK. The consequent rise in sMer coupled to reduced expression of MerTK impairs binding and internalization of POS in ARPE-19 cells.

Gene Correction Recovers Phagocytosis in Retinal Pigment Epithelium Derived from Retinitis Pigmentosa-Human-Induced Pluripotent Stem Cells.

Hereditary retinal dystrophies (HRD) represent a significant cause of blindness, affecting mostly retinal pigment epithelium (RPE) and photoreceptors (PRs), and currently suffer from a lack of effective treatments. Highly specialized RPE and PR cells interact mutually in the functional retina, therefore primary HRD affecting one cell type leading to a secondary HRD in the other cells. Phagocytosis is one of the primary functions of the RPE and studies have discovered that mutations in the phagocytosis-associated gene Mer tyrosine kinase receptor (MERTK) lead to primary RPE dystrophy. Treatment strategies for this rare disease include the replacement of diseased RPE with healthy autologous RPE to prevent PR degeneration. The generation and directed differentiation of patient-derived human-induced pluripotent stem cells (hiPSCs) may provide a means to generate autologous therapeutically-relevant adult cells, including RPE and PR. However, the continued presence of the MERTK gene mutation in patient-derived hiPSCs represents a significant drawback. Recently, we reported the generation of a hiPSC model of MERTK-associated Retinitis Pigmentosa (RP) that recapitulates disease phenotype and the subsequent creation of gene-corrected RP-hiPSCs using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9. In this study, we differentiated gene-corrected RP-hiPSCs into RPE and found that these cells had recovered both wild-type MERTK protein expression and the lost phagocytosis of fluorescently-labeled photoreceptor outer segments observed in uncorrected RP-hiPSC-RPE. These findings provide proof-of-principle for the utility of gene-corrected hiPSCs as an unlimited cell source for personalized cell therapy of rare vision disorders.

Deterioration of phagocytosis in induced pluripotent stem cell-derived retinal pigment epithelial cells established from patients with retinitis pigmentosa carrying Mer tyrosine kinase mutations.

Retinitis pigmentosa (RP) is an incurable retinal degenerative disease with an unknown mechanism of disease progression. Mer tyrosine kinase (MERTK), which encodes a receptor of the Tyro3/Axl/Mer family of tyrosine kinases, is one of the causal genes of RP. MERTK is reportedly expressed in the retinal pigment epithelium (RPE) and is essential for phagocytosis of the photoreceptor outer segment. Here, we established induced pluripotent stem cells (iPSC) from patients with RP having homozygous or compound heterozygous mutations in MERTK, and from healthy subjects; the RP patient- and healthy control-derived iPSCs were differentiated into RPE cells. Although cytoskeleton staining suggested that polarity may have been disturbed mildly, there were no apparent morphological differences between the diseased and normal RPE cells. The internalization of photoreceptor outer segments in diseased iPSC-RPE cells was significantly lower than that in normal iPSC-RPE cells. This in vitro disease model may be useful for elucidating the mechanisms of disease progression and screening treatments for the disease.

The diterpene Manool extracted from Salvia tingitana lowers free radical production in retinal rod outer segments by inhibiting the extramitochondrial F1 Fo ATP synthase.

Uncontrolled oxidative stress production, especially in the outer retina is one of the causes of retinal degenerations. Mitochondria are considered the principal source of oxidative stress. However, a Reactive Oxygen Intermediates (ROI) production in the retinal photoreceptor layer seems to depend also on the expression of an extramitochondrial oxidative phosphorylation (OxPhos) machinery in the rod outer segments (OS). In fact, OS conduct aerobic metabolism, producing ATP through oxygen consumption, although it is devoid of mitochondria. As diterpenes display an antioxidant effect, we have evaluated the effect Manool, extracted from Salvia tingitana, on the extramitochondrial OxPhos and the ROI production in the retinal rod OS. Results confirm that the OxPhos machinery is ectopically expressed in the OS and that F1 Fo -ATP synthase is a target of Manool, which inhibited the OS ATP synthesis, binding the F1 moiety with high affinity, as analysed by molecular docking. Moreover, the overall slowdown of OxPhos metabolism reduced the ROI production elicited in the OS by light exposure, in vitro. In conclusion, data are consistent with the antioxidant properties of Salvia spp., suggesting its ability to lower oxidative stress production, a primary risk factor for degenerative retinal diseases. SIGNIFICANCE OF THE STUDY: Here we show that Manool, a diterpene extracted from Salvia tingitana has the potential to lower the free radical production by light-exposed rod outer segments in vitro, by specifically targeting the rod OS F1 Fo -ATP synthase belonging to the extramitochondrial OxPhos expressed on the disk membrane. The chosen experimental model allowed to show that the rod OS is a primary producer of oxidative stress linked to the pathogenesis of degenerative retinal diseases. Data are also consistent with the antioxidant and anti-inflammatory action of Salvia spp., suggesting a beneficial effect also in vivo.

Phagocytosis by the Retinal Pigment Epithelium: Recognition, Resolution, Recycling.

Tissue-resident phagocytes are responsible for the routine binding, engulfment, and resolution of their meals. Such populations of cells express appropriate surface receptors that are tailored to recognize the phagocytic targets of their niche and initiate the actin polymerization that drives internalization. Tissue-resident phagocytes also harbor enzymes and transporters along the endocytic pathway that orchestrate the resolution of ingested macromolecules from the phagolysosome. Solutes fluxed from the endocytic pathway and into the cytosol can then be reutilized by the phagocyte or exported for their use by neighboring cells. Such a fundamental metabolic coupling between resident phagocytes and the tissue in which they reside is well-emphasized in the case of retinal pigment epithelial (RPE) cells; specialized phagocytes that are responsible for the turnover of photoreceptor outer segments (POS). Photoreceptors are prone to photo-oxidative damage and their long-term health depends enormously on the disposal of aged portions of the outer segment. The phagocytosis of the POS by the RPE is the sole means of this turnover and clearance. RPE are themselves mitotically quiescent and therefore must resolve the ingested material to prevent their toxic accumulation in the lysosome that otherwise leads to retinal disorders. Here we describe the sequence of events underlying the healthy turnover of photoreceptors by the RPE with an emphasis on the signaling that ensures the phagocytosis of the distal POS and on the transport of solutes from the phagosome that supersedes its resolution. While other systems may utilize different receptors and transporters, the biophysical and metabolic manifestations of such events are expected to apply to all tissue-resident phagocytes that perform regular phagocytic programs.

KCNJ13 Gene Deletion Impairs Cell Alignment and Phagocytosis in Retinal Pigment Epithelium Derived from Human-Induced Pluripotent Stem Cells.

Purpose: The purpose of this study was to establish and analyze a cell model of Leber congenital amaurosis type 16 (LCA16), which is caused by mutations in the KCNJ13 gene encoding Kir7.1, an inward-rectifying potassium ion channel. Methods: The two guide RNAs specific to the target sites in the KCNJ13 gene were designed and KCNJ13 knock-out (KO) human-induced pluripotent stem cells (hiPSCs) were generated using the CRISPR/Cas9 system. The KCNJ13-KO hiPSCs were differentiated into retinal pigment epithelial cells (hiPSC-RPEs). The KCNJ13-KO in hiPSC-RPEs was confirmed by immunostaining. Phagocytic activity of hiPSC-RPEs was assessed using the uptake of fluorescently labeled porcine photoreceptor outer segments (POSs). Phagocytosis-related genes in RPE cells were assessed by quantitative polymerase chain reaction. Results: Most of the translated region of the KCNJ13 gene was deleted in the KCNJ13-KO hiPSCs by the CRISPR/Cas9 system, and this confirmed that the Kir7.1 protein was not present in RPE cells induced from the hiPSCs. Expression of RPE marker genes such as BEST1 and CRALBP was retained in the wild-type (WT) and in the KCNJ13-KO hiPSC-RPE cells. However, phagocytic activity and expression of phagocytosis-related genes in the KCNJ13-null hiPSC-RPE cells were significantly reduced compared to those of WT. Conclusions: We succeeded in generating an RPE model of LCA16 using hiPSCs. We suggest that Kir7.1 is required for phagocytosis of POSs by RPE cells and that impaired phagocytosis in the absence of Kir7.1 would be involved in the retinal degeneration found in LCA16.

Potential Role of Myeloid-Derived Suppressor Cells (MDSCs) in Age-Related Macular Degeneration (AMD).

Myeloid cells, such as granulocytes/neutrophils and macrophages, have responsibilities that include pathogen destruction, waste material degradation, or antigen presentation upon inflammation. During persistent stress, myeloid cells can remain partially differentiated and adopt immunosuppressive functions. Myeloid-derived suppressor cells (MDSCs) are primarily beneficial upon restoring homeostasis after inflammation. Because of their ability to suppress adaptive immunity, MDSCs can also ameliorate autoimmune diseases and semi-allogenic responses, e.g., in pregnancy or transplantation. However, immunosuppression is not always desirable. In certain conditions, such as cancer or chronically inflamed tissue, MDSCs prevent restorative immune responses and thereby aggravate disease progression. Age-related macular degeneration (AMD) is the most common disease in Western countries that severely threatens the central vision of aged people. The pathogenesis of this multifactorial disease is not fully elucidated, but inflammation is known to participate in both dry and wet AMD. In this paper, we provide an overview about the potential role of MDSCs in the pathogenesis of AMD.

Photopigment Bleaching Phenomenon With Scanning Laser Ophthalmoscopy and Fluorescein Angiography.

Photopigment bleaching occurs with saturation of photoreceptor pigment by short-wavelength fundus autofluorescence imaging. This phenomenon is seen as characteristic hyperautofluorescence with subsequent imaging acquisition. Herein, a patient with multiple sclerosis was found to exhibit increased choroidal hyperfluorescence during fluorescein angiography (FA) that corresponded with a circumscribed area of intense blue light exposure during initial scanning laser ophthalmoscopy. To the authors' knowledge, this case is the first description of photobleaching phenomenon during FA and should be recognized as nonpathologic by the clinician. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:590-592.].

Oligomerization of Prph2 and Rom1 is essential for photoreceptor outer segment formation.

Mutations in peripherin 2 (PRPH2, also known as Rds), a tetraspanin protein found in photoreceptor outer segments (OSs), cause retinal degeneration ranging from rod-dominant retinitis pigmentosa (RP) to cone-dominant macular dystrophy (MD). Understanding why some Prph2 mutants affect rods while others affect cones remains a critical unanswered question. Prph2 is essential for OS structure and function and exhibits a very specific pattern of oligomerization with its homolog Rom1. Non-covalent Prph2/Rom1 homo- and hetero-tetramers assemble into higher-order covalently linked complexes held together by an intermolecular disulfide bond at Prph2-C150/Rom1-C153. Here we disrupt this crucial bond using a C150S-Prph2 knockin mouse line to study the role of Prph2 higher-order complex formation. We find that C150S-Prph2 traffics to the OS, interacts with Rom1 and forms non-covalent tetramers, but alone cannot support normal OS structure and function. However, C150S-Prph2 supports the initiation or elaboration of OS disc structures, and improves rod OS ultrastructure in the presence of wild-type (WT) Prph2 (i.e. Prph2C150S/+ versus Prph2+/-). Prph2C150S/+ animals exhibit haploinsufficiency in rods, but a dominant-negative phenotype in cones, suggesting cones have a different requirement for large Prph2 complexes than rods. Importantly, cone but not rod function can be improved by the addition of one Prph2Y141C allele, a mutation responsible for pattern dystrophy owing to the extra cysteine. Combined these findings show that covalently linked Prph2 complexes are essential for OS formation, but not for Prph2 targeting to the OS, and that cones are especially sensitive to having a broad distribution of Prph2 complex types (i.e. tetramers and large complexes).

Usherin defects lead to early-onset retinal dysfunction in zebrafish.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

Glucosamine-Induced Autophagy through AMPK⁻mTOR Pathway Attenuates Lipofuscin-Like Autofluorescence in Human Retinal Pigment Epithelial Cells In Vitro.

Age-related macular degeneration (AMD) is a vision-threatening age-associated disease. The retinal pigment epithelial (RPE) cells phagocytose and digest photoreceptor outer segment (POS). Incomplete digestion of POS leads to lipofuscin accumulation, which contributes to the pathology of the AMD. Autophagy could help reduce the amount of lipofuscin accumulation. In the present study, we evaluated the effects of glucosamine (GlcN), a natural supplement, on the induction of autophagy and POS-derived lipofuscin-like autofluorescence (LLAF) in ARPE-19 cells in vitro, and investigated the potential molecular pathway involved. Our results revealed that GlcN had no effect on phagocytosis of POS at the lower doses. GlcN treatment induced autophagy in cells. GlcN decreased the LLAF in native POS-treated cells, whereas malondialdehyde or 4-hydroxynonenal-modified POS attenuated this effect. 3-Methyladenine inhibited GlcN-induced autophagy and attenuated the effect of GlcN on the decrease of the native POS-derived LLAF. Furthermore, GlcN induced the phosphorylation of AMP-activated protein kinase (AMPK) and inhibited the phosphorylation of mammalian target of rapamycin (mTOR), whereas Compound C inhibited these effects of GlcN. Altogether, these results suggest that GlcN decreased the native POS-derived LLAF through induction of autophagy, at least in part, by the AMPK⁻mTOR pathway. This mechanism has potential for the preventive treatment of lipofuscin-related retinal degeneration such as AMD.

Cell Culture Analysis of the Phagocytosis of Photoreceptor Outer Segments by Primary Mouse RPE Cells.

The phagocytosis of photoreceptor outer segments (POSs) by the retinal pigment epithelium (RPE) is essential for retinal homeostasis. Defects in this process can be caused by mutations in the photoreceptor cells, the RPE cells, or both cell types. This function can be experimentally investigated by performing an in vitro phagocytosis assay, in which cultured RPE cells are challenged with isolated POSs, and subsequently tested for their ability to degrade the POSs. A significant advantage of this approach is that mutant phenotypes can be attributed either to the photoreceptor or the RPE cells, by experimenting with different permutations of mutant and control photoreceptor and RPE cells. In this chapter, we detail the method for a double-immunofluorescence assay for analysis of the binding, ingestion, and subsequent degradation of isolated mouse POSs by cultured mouse primary RPE cells.

Human L- and M-opsins restore M-cone function in a mouse model for human blue cone monochromacy.

Purpose: Blue cone monochromacy (BCM) is an X-linked congenital vision disorder characterized by complete loss or severely reduced L- and M-cone function. Patients with BCM display poor visual acuity, severely impaired color discrimination, myopia, nystagmus, and minimally detectable cone-mediated electroretinogram. Recent studies of patients with BCM with adaptive optics scanning laser ophthalmoscopy (AOSLO) showed that they have a disrupted cone mosaic with reduced numbers of cones in the fovea that is normally dominated by L- and M-cones. The remaining cones in the fovea have significantly shortened outer segments but retain sufficient structural integrity to serve as potential gene therapy targets. In this study, we tested whether exogenously expressed human L- and M-opsins can rescue M-cone function in an M-opsin knockout (Opn1mw-/- ) mouse model for BCM. Methods: Adeno-associated virus type 5 (AAV5) vectors expressing OPN1LW, OPN1MW, or C-terminal tagged OPN1LW-Myc, or OPN1MW-HA driven by a cone-specific promoter were injected subretinally into one eye of Opn1mw-/- mice, while the contralateral eye served as the uninjected control. Expression of cone pigments was determined with western blotting and their cellular localization identified with immunohistochemistry. M-cone function was analyzed with electroretinogram (ERG). Antibodies against cone phototransduction proteins were used to study cone outer segment (OS) morphology in untreated and treated Opn1mw-/- eyes. Results: We showed that cones in the dorsal retina of the Opn1mw-/- mouse do not form outer segments, resembling cones that lack outer segments in the human BCM fovea. We further showed that AAV5-mediated expression of either human M- or L-opsin individually or combined promotes regrowth of cone outer segments and rescues M-cone function in the treated Opn1mw-/- dorsal retina. Conclusions: Exogenously expressed human opsins can regenerate cone outer segments and rescue M-cone function in Opn1mw-/- mice, thus providing a proof-of-concept gene therapy in an animal model of BCM.

Regulated efflux of photoreceptor outer segment-derived cholesterol by human RPE cells.

Genetic studies have linked age-related macular degeneration (AMD) to genes involved in high-density lipoprotein (HDL) metabolism, including ATP-binding cassette transporter A1 (ABCA1). The retinal pigment epithelium (RPE) handles large amounts of lipids, among others cholesterol, partially derived from internalized photoreceptor outer segments (OS) and lipids physiologically accumulate in the aging eye. To analyze the potential function of ABCA1 in the eye, we measured cholesterol efflux, the first step of HDL generation, in RPE cells. We show the expression of selected genes related to HDL metabolism in mouse and human eyecups as well as in ARPE-19 and human primary RPE cells. Immunofluorescence staining revealed localization of ABCA1 on both sides of polarized RPE cells. This was functionally confirmed by directional efflux to apolipoprotein AI (ApoA-I) of 3H-labeled cholesterol given to the cells via serum or via OS. ABCA1 expression and activity was modulated using a liver-X-receptor (LXR) agonist and an ABCA1 neutralizing antibody, demonstrating that the efflux was ABCA1-dependent. We concluded that the ABCA1-mediated lipid efflux pathway, and hence HDL biosynthesis, is functional in RPE cells towards both the basal (choroidal) and apical (subretinal) space. Impaired activity of the pathway might cause age-related perturbations of lipid homeostasis in the outer retina and thus may contribute to disease development and/or progression.

RUBCN/rubicon and EGFR regulate lysosomal degradative processes in the retinal pigment epithelium (RPE) of the eye.

Macroautophagy/autophagy is an intracellular stress survival and recycling system whereas phagocytosis internalizes material from the extracellular milieu; yet, both pathways utilize lysosomes for cargo degradation. Whereas autophagy occurs in all cells, phagocytosis is performed by cell types such as macrophages and the retinal pigment epithelial (RPE) cells of the eye where it is supported by the noncanonical autophagy process termed LC3-associated phagocytosis (LAP). Autophagy and LAP are distinct pathways that use many of the same mediators and must compete for cellular resources, suggesting that cells may regulate both processes under homeostatic and stress conditions. Our data reveal that RPE cells promote LAP through the expression of RUBCN/Rubicon (RUN domain and cysteine-rich domain containing Beclin 1-interacting protein) and suppress autophagy through the activation of EGFR (epidermal growth factor receptor). In the morning when photoreceptor outer segments (POS) phagocytosis and LAP are highest, RUBCN expression is increased. At the same time, outer segment phagocytosis activates the EGFR resulting in MTOR (mechanistic target of rapamycin [serine/threonine kinase]) stimulation, the accumulation of SQSTM1/p62, and the phosphorylation of BECN1 (Beclin 1, autophagy related) on an inhibitory residue thereby suppressing autophagy. Silencing Rubcn, preventing EGFR activity or directly inducing autophagy in RPE cells by starvation inhibits phagocytic degradation of POS. Thus, RPE cells regulate lysosomal pathways during the critical period of POS phagocytosis to support retinal homeostasis.

Mertk gene expression and photoreceptor outer segment phagocytosis by cultured rat bone marrow mesenchymal stem cells.

BACKGROUND: Bone marrow mesenchymal stem cells (BM-MSCs) are multipotential stem cells that have been used for a broad spectrum of indications. Several investigations have used BM-MSCs to promote photoreceptor survival and suggested that BM-MSCs are a potential source of cell replacement therapy for some forms of retinal degeneration. PURPOSE: To investigate the expression of the MER proto-oncogene, tyrosine kinase (Mertk), involved in the disruption of RPE phagocytosis and the onset of autosomal recessive retinitis pigmentosa in rat BM-MSCs and to compare phagocytosis of the photoreceptor outer segment (POS) by BM-MSCs and RPE cells in vitro. METHODS: MSCs were isolated from the bone marrow of Brown Norway rats. Reverse transcription-PCR (RT-PCR) and western blot analyses were used to examine the expression of Mertk. The phagocytized POS was detected with double fluorescent labeling, transmission electron microscopy, and scanning electron microscopy. RESULTS: Mertk expression did not differ among the first three passages of BM-MSCs. Mertk gene expression was greater in the BM-MSCs than the RPE cells. Mertk protein expression in the BM-MSCs was similar to that in the RPE cells in the primary passage and was greater than that in the RPE cells in the other two passages. BM-MSCs at the first three passages phagocytized the POS more strongly than the RPE cells. The process of BM-MSC phagocytosis was similar to that of the RPE cells. CONCLUSIONS: BM-MSCs may be an effective cell source for treating retinal degeneration in terms of phagocytosis of the POS.

The Ciliopathy Gene ahi1 Is Required for Zebrafish Cone Photoreceptor Outer Segment Morphogenesis and Survival.

Purpose: Joubert syndrome (JBTS) is an autosomal recessive ciliopathy with considerable phenotypic variability. In addition to central nervous system abnormalities, a subset of JBTS patients exhibit retinal dystrophy and/or kidney disease. Mutations in the AHI1 gene are causative for approximately 10% of all JBTS cases. The purpose of this study was to generate ahi1 mutant alleles in zebrafish and to characterize the retinal phenotypes. Methods: Zebrafish ahi1 mutants were generated using transcription activator-like effector nucleases (TALENs). Expression analysis was performed by whole-mount in situ hybridization. Anatomic and molecular characterization of photoreceptors was investigated by histology, electron microscopy, and immunohistochemistry. The optokinetic response (OKR) behavior assay was used to assess visual function. Kidney cilia were evaluated by whole-mount immunostaining. Results: The ahi1lri46 mutation in zebrafish resulted in shorter cone outer segments but did not affect visual behavior at 5 days after fertilization (dpf). No defects in rod morphology or rhodopsin localization were observed at 5 dpf. By 5 months of age, cone degeneration and rhodopsin mislocalization in rod photoreceptors was observed. The connecting cilium formed normally and Cc2d2a and Cep290 localized properly. Distal pronephric duct cilia were absent in mutant fish; however, only 9% of ahi1 mutants had kidney cysts by 5 dpf, suggesting that the pronephros remained largely functional. Conclusions: The results indicate that Ahi1 is required for photoreceptor disc morphogenesis and outer segment maintenance in zebrafish.

The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.

The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H+ -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding ßA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression.

Gli1 Expression in Human Epiretinal Membranes.

Purpose: We evaluate the expression of Gli1 in human epiretinal membranes (ERM) and correlate this with clinical data. Methods: We prospectively recruited patients with ERM. A total of 33 human ERM specimens were immunolabeled with anti-Gli1 antibody and the number of total cells/hyperfield (HF), Gli1(+) cells/HF, and the percentage of Gli1(+) cells/total cells were calculated. We evaluated the interrelationship of cellular properties and clinical findings, such as presence of diabetic retinopathy (DR), retinal breaks, intraocular inflammation, central foveal thickness, maximal retinal thickness, retinal contraction, lamellar holes, pseudoholes, the attenuation or absence of an inner segment/outer segment (IS/OS) junction/external limiting membrane (ELM), cystic changes, and paravascular inner retinal defects. Results: Among 33 specimens, 25 specimens (75.8%) showed nuclear Gli1 expression. The mean Gli1(+) cells/total cells was 54.0 ± 36.7% (range, 0%-92.8%). There was significantly higher expression of Gli1(+) cells in ERM specimens from patients with DR (P = 0.014), and lower expression from patients with retinal breaks (P = 0.022). Epiretinal membrane specimens from patients with alteration of IS/OS junction/ELM or cystic changes on OCT showed higher percentage of Gli1(+) cells/total cells. Conclusions: Gli1 expression was detected in most ERM specimens. Patients who had DR or OCT findings indicating chronic retinal insults showed higher Gli1 expression. Gli1 may have a role in the pathogenesis of ERM after chronic retinal insults.

Abnormal photoreceptor outer segment development and early retinal degeneration in kif3a mutant zebrafish.

Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A (kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport.