Genetic LAMP2 deficiency accelerates the age-associated formation of basal laminar deposits in the retina.

The early stages of age-related macular degeneration (AMD) are characterized by the accumulation of basal laminar deposits (BLamDs). The mechanism for BLamDs accumulating between the retinal pigment epithelium (RPE) and its basal lamina remains elusive. Here we examined the role in AMD of lysosome-associated membrane protein-2 (LAMP2), a glycoprotein that plays a critical role in lysosomal biogenesis and maturation of autophagosomes/phagosomes. LAMP2 was preferentially expressed by RPE cells, and its expression declined with age. Deletion of the Lamp2 gene in mice resulted in age-dependent autofluorescence abnormalities of the fundus, thickening of Bruch's membrane, and the formation of BLamDs, resembling histopathological changes occurring in AMD. Moreover, LAMP2-deficient mice developed molecular signatures similar to those found in human AMD-namely, the accumulation of APOE, APOA1, clusterin, and vitronectin-adjacent to BLamDs. In contrast, collagen 4, laminin, and fibronectin, which are extracellular matrix proteins constituting RPE basal lamina and Bruch's membrane were reduced in Lamp2 knockout (KO) mice. Mechanistically, retarded phagocytic degradation of photoreceptor outer segments compromised lysosomal degradation and increased exocytosis in LAMP2-deficient RPE cells. The accumulation of BLamDs observed in LAMP2-deficient mice was eventually followed by loss of the RPE and photoreceptors. Finally, we observed loss of LAMP2 expression along with ultramicroscopic features of abnormal phagocytosis and exocytosis in eyes from AMD patients but not from control individuals. Taken together, these results indicate an important role for LAMP2 in RPE function in health and disease, suggesting that LAMP2 reduction may contribute to the formation of BLamDs in AMD.

Birt-Hogg-Dubé syndrome associated with chorioretinopathy and nyctalopia: a case report and review of the literature.

PURPOSE: To report a rare case of Birt-Hogg-Dubé Syndrome (BHD) with progressive chorioretinopathy. METHODS: Case report. RESULTS: A 55-year-old woman presented with longstanding nyctalopia attributed to a congenital retinal dystrophy, but no prior genetic testing. Her posterior pole examination demonstrated retinal pigment epithelium (RPE) mottling with extensive macular drusen and paracentral chorioretinal atrophy, consistent with a fleck retinopathy. Her past medical history was remarkable for nephrectomy for unilateral renal malignancy, parotid tumors and thyroid nodules. Dark adaptation time was prolonged, and electroretinography (ERG) revealed abnormal waveforms with depressed amplitudes. Genetic testing confirmed a deletion mutation in the folliculin (FLCN) gene and was negative for other relevant mutations, including EFEMP1 responsible for autosomal dominant macular and peripapillary drusen in Doyne honeycomb retinal dystrophy and TIMP3 responsible for Sorsby Fundus Dystrophy. CONCLUSION: BHD is a rare autosomal-dominant disorder with multi-systemic clinical manifestations caused by a mutation in the FLCN gene. Affected individuals are prone to renal and pulmonary cysts, renal cancer, and fibrofolliculomas. Reports on ocular manifestations of BHD include eyelid fibrofolliculomas, flecked chorioretinopathy, choroidal melanoma, choroidal melanoma with sector melanocytosis, and retinal pigment epithelial micro-detachments. In this case of BHD, we note a fleck retinopathy with bilateral chorioretinal atrophy, displaying a phenotype of extensive chorioretinopathy associated with impaired dark adaptation and ERG abnormalities. ABBREVIATIONS: BHD: Birt-Hogg-Dubé syndrome; FLCN: Folliculin. RPE: retinal pigment epithelium; OD: Oculus dexter (right eye); OS: Oculus sinister (left eye). OU: Oculus uterque (both eyes); ERG: electroretinogram; mfERG: multifocal electroretinography. ffERG: full-field electroretinography; FAF: fundus autofluorescence; OCT: optical coherence tomography; FA: fluorescein angiography; DA: dark-adapted; LA: light-adapted; mTOR: mammalian target of rapamycin; EFEMP1: epithelial growth factor-containing fibulin-like extracellular matrix protein 1; VPS13B: Vacuolar Protein Sorting 13 Homolog B; AGBL5: AATP/GTP-Binding Protein Like 5; ALMS1: Alstrom Syndrome 1; COL1BA1: Collagen Type I Beta, Alpha Chain 1; PDE6A: Rod Phosphodiesterase 6-alpha; USH2A: Usherin 2a; VCAN: Versican; RP: Retinitis pigmentosa; AR: Autosomal recessive.

Choroidal amelanotic mass with vermiform margins, vision loss, and extreme fatigue.

PURPOSE: To describe a case of a previously healthy middle-aged male with an atypical choroidal mass and fatigue. METHODS: Case report. RESULTS: A 56-year-old Caucasian male presented with decreased vision in the right eye (OD) and a choroidal amelanotic mass OD with subretinal fluid. He received bevacizumab (1.25 mg/0.05 mL) injections by multiple providers without improvement in vision and with tumor progression. Visual acuity was 20/400 OD and 20/25 left eye (OS). The anterior segment was unremarkable in both eyes (OU). He was referred with suspected diagnosis of choroidal melanoma, metastasis, or osteoma. On examination, a choroidal amelanotic tumor with vermiform margins measuring 12 mm in base and 1.8 mm in thickness was seen OD. Smaller satellite lesions were noted inferiorly. Fundus OS was normal. Infectious evaluation was unrevealing and angiotensin-converting enzyme, lysozyme, and chest x-ray were normal. Fine-needle aspiration biopsy was declined by the patient. Given the clinical suspicion of choroidal sarcoidosis with characteristic tumor color, vermiform margins, and presence of satellite lesions, oral corticosteroids 60 mg/day for 2 months were started, followed by sub-Tenons triamcinolone acetonide (40mg/1 mL) injection three months later and urgent rheumatologic evaluation. He self-discontinued the oral corticosteroids and the tumor progressed by the next follow-up. Oral corticosteroids were restarted at 80mg/day. 16 months after his presentation to us, he experienced fatigue with electrocardiogram findings of third-degree heart block from sarcoidosis. He received an intracardiac defibrillator and 80 mg/day of corticosteroids with plans to initiate methotrexate for stronger immunosuppression. CONCLUSION: Understanding the numerous manifestations of ocular and systemic sarcoidosis is crucial. Choroidal sarcoidosis characteristically exhibits vermiform margins. Close follow-up and systemic monitoring is necessary in patients presenting with ocular signs of sarcoidosis. PRECIS: The diagnosis of choroidal sarcoidosis can be challenging. Features including vermiform tumor margins, satellite lesions, and choroidal infiltration on OCT are suggestive of choroidal sarcoidosis. All patients with ocular sarcoidosis should be monitored for systemic involvement. In this case, the ocular manifestation guided the diagnosis and eventually the treatment of cardiac sarcoidosis.

Bilateral Exudative Retinal Detachments and Panuveitis in a Patient with Multiple Myeloma.

PURPOSE: To report a case of bilateral exudative retinal detachments and panuveitis in a patient with multiple myeloma (MM). CASE REPORT: A 54-year-old patient with non-proliferative diabetic retinopathy was referred with blurred vision and scotomas in both eyes (OU). Three months prior to the onset of ocular symptoms, he was diagnosed with systemic MM and was receiving chemotherapy. Clinical examination revealed best-corrected visual acuities of 20/80 OU, rare anterior chamber cell, 2+ vitreous cell, diffuse intraretinal hemorrhages, and exudative retinal detachments (RD). Optical coherence tomography of the macula showed central subretinal fluid with cystic intraretinal fluid OU. The findings were consistent with panuveitis and exudative RD in the setting of MM. He reported symptomatic improvement after plasmapheresis and oral prednisone initiation. CONCLUSION: Extensive, bilateral exudative RD and panuveitis are rare but potentially sight-threatening findings in patients with MM.

Targeting the YAP/TAZ Pathway in Uveal and Conjunctival Melanoma With Verteporfin.

Purpose: The purpose of this study was to determine whether YAP/TAZ activation in uveal melanoma (UM) and the susceptibility of melanoma cell lines to YAP/TAZ inhibition by verteporfin (VP) is related to the tumor's genetic background. Methods: Characteristics of 144 patients with enucleated UM were analyzed together with mRNA expression levels of YAP/TAZ-related genes (80 patients from the The Cancer Genome Atlas [TCGA] project and 64 patients from Leiden, The Netherlands). VP was administered to cell lines 92.1, OMM1, Mel270, XMP46, and MM28 (UM), CRMM1 and CRMM2 (conjunctival melanoma), and OCM3 (cutaneous melanoma). Viability, growth speed, and expression of YAP1-related proteins were assessed. Results: In TCGA data, high expression of YAP1 and WWTR1 correlated with the presence of monosomy 3 (P = 0.009 and P < 0.001, respectively) and BAP1-loss (P = 0.003 and P = 0.001, respectively) in the primary UM; metastasis development correlated with higher expression of YAP1 (P = 0.05) and WWTR1 (P = 0.003). In Leiden data, downstream transcription factor TEAD4 was increased in cases with M3/BAP1-loss (P = 0.002 and P = 0.006) and related to metastasis (P = 0.004). UM cell lines 92.1, OMM1, and Mel270 (GNAQ/11-mutation, BAP1-positive) and the fast-growing cell line OCM3 (BRAF-mutation) showed decreased proliferation after exposure to VP. Two slow-growing UM cell lines XMP46 and MM28 (GNAQ/11-mutation, BAP1-negative) were not sensitive to VP, and neither were the two conjunctival melanoma cell lines (BRAF/NRAS-mutation). Conclusions: High risk UM showed an increased expression of YAP/TAZ-related genes. Although most UM cell lines responded in vitro to VP, BAP1-negative and conjunctival melanoma cell lines did not. Not only the mutational background, but also cell growth rate is an important predictor of response to YAP/TAZ inhibition by VP.

Retinal Imaging Using a Confocal Scanning Laser Ophthalmoscope-Based High-Magnification Module.

PURPOSE: To evaluate the usefulness of a high-magnification module (HMM) lens to visualize retinal photoreceptors, retinal nerve fiber layer (RNFL), and superficial retinal vasculature in physiologic and pathologic retinal conditions. DESIGN: Observational descriptive study. PARTICIPANTS: Thirty-two participants with normal and pathologic retina examination results. METHODS: Normal and pathologic maculae were imaged in vivo using still and video HMM lens modes, with fixation and contrast adjustments to enhance visualization. The HMM images were classified qualitatively based on structures identified as either good (photoreceptors seen), average (photoreceptor mosaic cannot be visualized clearly, retinal vessels and other retinal changes can be seen), or poor (no identifiable structures). Selected eyes were imaged with fundus photography, OCT, OCT angiography, indocyanine green angiography, and fluorescein angiography for comparison with the pathologic maculae. MAIN OUTCOMES MEASURES: Description of HMM module-obtained macula images. RESULTS: From 32 eyes imaged (16 normal and 16 pathologic retinas), 12 of 16 normal and 11 of 16 pathologic retinas demonstrated at least average image quality, in which retinal vasculature and landmarks could be visualized. The mosaic pattern of hexagonal shapes representing photoreceptors could not be resolved in most pathologic retinas. For the retinas in which the photoreceptor mosaics were visualized (12 of 16 normal and 2 of 16 pathologic retinas), parafoveal mosaic patterns appeared denser with better image quality for all participants compared with foveal photoreceptors. Difficulty in resolving the photoreceptors in the umbo, fovea, and perifovea was encountered, similar to what has been reported with adaptive optics devices. The RNFL was seen as arcuate hyperreflective bundles. Flow was observed in the macular microvasculature. Poorly resolved photoreceptors and scattered hyperreflective foci were correlated with changes in the retinal pigment epithelium in eyes with age-related macular degeneration or central serous chorioretinopathy. Macular striae were seen in eyes with epiretinal membrane. CONCLUSIONS: In most eyes, regardless of whether retinal pathologic features were present, it was challenging to obtain average quality (or better) images. In the few participants with good-quality imaging, the parafoveal photoreceptor mosaic, vascular flow, and various features of pathologic eyes could be visualized.

Acadesine suppresses TNF-α induced complement component 3 (C3), in retinal pigment epithelial (RPE) cells.

RATIONALE: Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness in the developed world. Aging, inflammation and complement dysregulation affecting the retinal pigment epithelium (RPE), are considered significant contributors in its pathogenesis and several evidences have linked tumor necrosis factor alpha (TNF-α) and complement component 3 (C3) with AMD. Acadesine, an analog of AMP and an AMP-activated protein kinase (AMPK) activator, has been shown to have cytoprotective effects in human clinical trials as well as having anti-inflammatory and anti-vascular exudative effects in animals. The purpose of this study was to evaluate if acadesine is able to suppress TNF-α induced C3 in RPE cells. METHODS: ARPE-19 and human primary RPE cells were cultured and allowed to grow to confluence. TNF-α was used for C3 induction in the presence or absence of acadesine. Small molecule inhibitors and siRNA were used to determine if acadesine exerts its effect via the extracellular or intracellular pathway and to evaluate the importance of AMPK for these effects. The expression level of C3 was determined by immunoblot analysis. RESULTS: Acadesine suppresses TNF-α induced C3 in a dose dependent manner. When we utilized the adenosine receptor inhibitor dipyridamole (DPY) along with acadesine, acadesine's effects were abolished, indicating the necessity of acadesine to enter the cell in order to exert it's action. However, pretreatment with 5-iodotubericidin (5-Iodo), an adenosine kinase (AK) inhibitor, didn't prevent acadesine from decreasing TNF-α induced C3 expression suggesting that acadesine does not exert its effect through AMP conversion and subsequent activation of AMPK. Consistent with this, knockdown of AMPK α catalytic subunit did not affect the inhibitory effect of acadesine on TNF-α upregulation of C3. CONCLUSIONS: Our results suggest that acadesine suppresses TNF-α induced C3, likely through an AMPK-independent pathway, and could have potential use in complement over activation diseases.

Morphological changes in intraretinal microvascular abnormalities after anti-VEGF therapy visualized on optical coherence tomography angiography.

BACKGROUND: To examine the baseline morphological characteristics and alterations in intraretinal microvascular abnormalities (IRMAs) in response to anti-vascular endothelial growth factor (anti-VEGF) treatment, documented by optical coherence tomography angiography (OCTA) in diabetic eyes. METHODS: In this retrospective study, IRMAs were evaluated with multimodal imaging (fundus photography, fluorescein angiography, OCTA) in treatment-naïve diabetic eyes before and after anti-VEGF treatment for diabetic macular edema (DME) and/or proliferative diabetic retinopathy (PDR) and compared to diabetic control eyes with similar diabetic retinopathy (DR) severity that did not receive anti-VEGF therapy. The morphological characteristics of IRMAs on enface OCTA imaging were graded by masked readers at baseline, then after anti-VEGF therapy in treated eyes or after observation in control eyes. Characterization of interval changes in an IRMA were based on the following parameters: branching, vessel caliber and area of adjacent capillary non-perfusion. RESULTS: The treated group included 45 IRMA foci from 15 eyes of 11 patients, while the control group included 27 IRMA foci from 15 eyes of 14 patients. Following anti-VEGF treatment, enface OCTA demonstrated that 14 foci of IRMA (31%) demonstrated regression with normalization of appearance of the capillary bed, 20 IRMAs (44%) remained unchanged, six IRMAs (13%) progressed with enlargement or development of new IRMAs and five IRMAs (11%) demonstrated complete obliteration defined as IRMA disappearance with advancing capillary drop-out. In the control group, 17 IRMA (63%) remained stable, 8 IRMAs (29.6%) progressed and 2 experienced total obliteration (7.4%). The difference in rank order between the two groups was statistically significant (p = 0.022). CONCLUSIONS: In eyes with DR status post anti-VEGF therapy, foci of IRMAs have a variable course demonstrating one of four possible outcomes: regression, stability, progression or complete obliteration. In contrast, none of the untreated control diabetic eyes demonstrated regression of IRMAs, consistent with known progression of DR severity in high risk eyes. Morphologic evaluation of IRMAs with OCTA may help to monitor changes in retinal blood flow as well as the response to anti-VEGF treatment.

Repeatability and Reproducibility of Photoreceptor Density Measurement in the Macula Using the Spectralis High Magnification Module.

PURPOSE: To evaluate the repeatability and reproducibility of photoreceptor density assessment with manual cell counting in healthy participants imaged with the Heidelberg Spectralis High Magnification Module (HMM). DESIGN: Precision study, evaluation of diagnostic test or technology. PARTICIPANTS: Eleven eyes of 8 participants. METHODS: Images were acquired using the Spectralis HMM by a single operator during 2 separate imaging sessions. The 3 highest-quality images of each eye from each session were selected for analysis and coregistered. For a subset of participants, a second operator acquired images in 1 session, and images with the best quality were selected for analysis. Photoreceptor densities were obtained by manual counts in squares of 0.0625 mm2 located in the parafovea. Repeatability (intragrader and intrasession) and reproducibility (interoperator, intergrader, and intersession) were assessed by calculating the intraclass correlation coefficient (ICC) from linear mixed effects models. Bland-Altman plots, coefficients of repeatability, and Pearson correlation results were reported. MAIN OUTCOME MEASURES: Intragrader, intrasession, intersession, interoperator, and intergrader ICC estimates and their 95% confidence intervals for photoreceptor density measurements in the parafovea. RESULTS: Twenty-four eyes of 13 healthy participants were imaged initially. Of these, 11 eyes (45.83%) of 8 participants that had at least 3 acceptable images in each session were included in this study. Mean parafoveal photoreceptor density was 14 988 cells/mm2 (standard deviation, 1403.15 cells/mm2). Intragrader ICC was 0.84 (95% confidence interval, 0.57-0.95), intrasession ICC was 0.69 (95% confidence interval, 0.17-0.86), intersession ICC was 0.88 (95% confidence interval, 0.53-0.96), interoperator ICC was 0.70 (95% confidence interval, 0-0.95), and intergrader ICC was 0.22 (95% confidence interval, 0-0.71). CONCLUSIONS: Images obtained with the HMM allow for photoreceptor mosaic visualization in the macular area, mainly in the parafovea. Although densities obtained are in accordance with other reported methods in the literature, variability within and between images of the apparent cell mosaic were observed, and this study did not demonstrate high repeatability or reproducibility for quantitative assessments using the manual counting method.

Toxoplasma GRA15 Activates the NF-κB Pathway through Interactions with TNF Receptor-Associated Factors.

The protozoan parasite Toxoplasma gondii secretes proteins from specialized organelles, the rhoptries, and dense granules, which are involved in the modulation of host cell processes. Dense granule protein GRA15 activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. Exactly how GRA15 activates the NF-κB pathway is unknown. Here we show that GRA15 interacts with tumor necrosis factor receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. We identified several TRAF binding sites in the GRA15 amino acid sequence and showed that these are involved in NF-κB activation. Furthermore, a TRAF2 knockout cell line has impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.IMPORTANCE The parasite Toxoplasma can cause birth defects and severe disease in immunosuppressed patients. Strain differences in pathogenicity exist, and these differences are due to polymorphic effector proteins that Toxoplasma secretes into the host cell to coopt host cell functions. The effector protein GRA15 of some Toxoplasma strains activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. We show that GRA15 interacts with TNF receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. Deletion of TRAF-binding sites in GRA15 greatly reduces its ability to activate the NF-κB pathway, and TRAF2 knockout cells have impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.

Receptor interacting protein kinase 3 (RIP3) regulates iPSCs generation through modulating cell cycle progression genes.

The molecular mechanisms involved in induced pluripotent stem cells (iPSCs) generation are poorly understood. The cell death machinery of apoptosis-inducing caspases have been shown to facilitate the process of iPSCs reprogramming. However, the effect of other cell death processes, such as programmed necrosis (necroptosis), on iPSCs induction has not been studied. In this study, we investigated the role of receptor-interacting protein kinase 3 (RIP3), an essential regulator of necroptosis, in reprogramming mouse embryonic fibroblast cells (MEFs) into iPSCs. RIP3 was found to be upregulated in iPSCs compared to MEFs. Deletion of RIP3 dramatically suppressed the reprogramming of iPSCs (~82%). RNA-seq analysis and qRT-PCR showed that RIP3 KO MEFs expressed lower levels of genes that control cell cycle progression and cell division and higher levels of extracellular matrix-regulating genes. The growth rate of RIP3 KO MEFs was significantly slower than WT MEFs. These findings can partially explain the inhibitory effects of RIP3 deletion on iPSCs generation and show for the first time that the necroptosis kinase RIP3 plays an important role in iPSC reprogramming. In contrast to RIP3, the kinase and scaffolding functions of RIPK1 appeared to have distinct effects on reprogramming.

Global Analysis of Macular Choriocapillaris Perfusion in Dry Age-Related Macular Degeneration using Swept-Source Optical Coherence Tomography Angiography.

Purpose: Swept-source optical coherence tomography angiography (SS-OCTA) was used to investigate if the clinical stage of dry age-related macular degeneration (AMD) was correlated with global and regional macular choriocapillaris (CC) perfusion. Methods: In this retrospective, cross-sectional study, 6 × 6-mm SS-OCTA images from eyes with early, intermediate, and advanced dry AMD (56 eyes, 41 patients) were analyzed using algorithms described in the literature to assess regional flow deficit percentage (FD%) and average flow deficit size. Regions were defined by concentric areas centered on the fovea: a 1-mm-diameter area, 3-mm-diameter ring, 5-mm-diameter area, 5-mm-diameter ring, and 6 × 6-mm whole image. Data were modeled using the generalized estimating equations approach. Results: The relationship between age and CC FD% and average flow deficit size was statistically significant (P ≤ 0.05) in all regions of analysis by linear modeling. The relationship between dry AMD stage and FD% was statistically significant by linear modeling in the 5-mm ring, and between dry AMD stage and average flow deficit size in the 3-mm ring, 5-mm area, 5-mm ring, and 6 × 6-mm whole image. Conclusions: Linear modeling suggests a statistically significant relationship between dry AMD stage and CC perfusion, most prominent in the more peripheral regions of the macula.