Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins.

The human apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found, by examining cells stably expressing each APOE isoform, that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-Golgi compartment is degraded through autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells, and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that might contribute to AD pathogenesis. This article has an associated First Person interview with the first author of the paper.

RIPK necrotic cell death pathway in both donor photoreceptor and host immune cells synergize to affect photoreceptor graft survival.

Photoreceptor transplant has been put forward as a repair strategy to tackle degenerated retinas. Nonetheless, cell death and immune rejection seriously limit the success of this strategy, with only a small fraction of transplanted cells surviving. Improving the survival of transplanted cells is of critical importance. Recent evidence has identified receptor-interacting protein kinase 3 (RIPK3) as a molecular trigger controlling necroptotic cell death and inflammation. However, its role in photoreceptor transplantation and regenerative medicine has not been studied. We hypothesized that modulation of RIPK3 to address both cell death and immunity could have advantageous effects on photoreceptor survival. In a model of inherited retinal degeneration, deletion of RIPK3 in donor photoreceptor precursors significantly increases the survival of transplanted cells. Simultaneous RIPK3 deletion in donor photoreceptors and recipients maximizes graft survival. Lastly, to discern the role of RIPK3 in the host immune response, bone marrow transplant experiments demonstrated that peripheral immune cell RIPK3 deficiency is protective for both donor and host photoreceptor survival. Interestingly, this finding is independent of photoreceptor transplantation, as the peripheral protective effect is also observed in an additional retinal detachment photoreceptor degeneration model. Altogether, these results indicate that immunomodulatory and neuroprotective strategies targeting the RIPK3 pathway can aid regenerative therapies of photoreceptor transplantation.

Effects of newer-generation anti-diabetics on diabetic retinopathy: a critical review.

Diabetic retinopathy (DR) is the leading etiology of blindness in the working population of the USA. Its long-term management relies on effective glycemic control. Seven anti-diabetic classes have been introduced for patients with type 2 diabetes (T2D) in the past two decades, with different glucose-lowering and cardiovascular benefits. Yet, their effects specifically on DR have not been studied in detail. A systematic review of the literature was conducted to investigate this topic, focusing on the available clinical data for T2D. Published studies were evaluated based on their level of statistical evidence, as long as they incorporated at least one endpoint or adverse event pertaining to retinal health. Fifty nine articles met our inclusion criteria and were grouped per anti-diabetic class as follows: alpha-glucosidase inhibitors (1), peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists (8), amylin analogs (1), glucagon-like peptide-1 (GLP-1) receptor agonists (28), dipeptidyl peptidase 4 (DPP-4) inhibitors (9), and sodium glucose co-transporter-2 (SGLT-2) inhibitors (9), plus one retrospective study and two meta-analyses evaluating more than one of the aforementioned anti-diabetic categories. We also reviewed publicly-announced results of trials for the recently-introduced class of twincretins. The available data indicates that most drugs in the newer anti-diabetic classes are neutral to DR progression; however, there are subclasses differences in specific drugs and T2D populations. In particular, there is evidence suggesting there may be worse diabetic macular edema with PPAR-gamma agonists, potential slight DR worsening with semaglutide (GLP-1 receptor agonist), and potential slight increase in the incidence of retinal vein occlusion in elderly and patients with advanced kidney disease receiving SGLT-2 inhibitors. All these warrant further investigation. Longer follow-up and systematic assessment of at least one DR-related endpoint are highly recommended for all future trials in the T2D field, to ultimately address this topic.

Associations of quantitative contrast sensitivity with wide-field swept-source optical coherence tomography angiography in retinal vein occlusion.

PURPOSE: To investigate associations between contrast sensitivity (CS) and vascular metrics on wide-field swept-source optical coherence tomography angiography (WF-SS-OCTA) in patients with retinal vein occlusion (RVO). METHODS: This prospectively recruited, cross-sectional observational study included RVO patients who underwent quantitative CS function (qCSF) testing and WF-SS-OCTA using 3 × 3, 6 × 6, and 12 × 12 mm angiograms on the same day. The study measured several qCSF outcomes and WF-SS-OCTA vascular metrics, including vessel density (VD), vessel skeletonized density (VSD), and foveal avascular zone (FAZ). The data were analyzed using multivariable regression analysis controlling for age and central subfield thickness (CST). RESULTS: A total of 43 RVO eyes of 43 patients and 30 fellow eyes were included. In RVO eyes, multiple vascular metrics were associated with CS outcomes but not visual acuity (VA). On 12 × 12 images, CS thresholds at 1 cpd, 1.5 cpd, and 3 cpd were significantly associated with VD and VSD, but VA was not. When comparing standardized regression coefficients, we found that vascular metrics had a larger effect size on CS than on VA. For instance, the standardized beta coefficient for FAZ area and CS at 6 cpd (ß* = - 0.46, p = 0.007) was larger than logMAR VA (ß* = 0.40, p = 0.011). CONCLUSION: Microvascular changes on WF-SS-OCTA in RVO had a larger effect size on CS than VA. This suggests CS may better reflect the microvascular changes of RVO compared to VA. qCSF-measured CS might be a valuable adjunct functional metric in evaluating RVO patients.

Evolution of macular atrophy in eyes with neovascular age-related macular degeneration compared to fellow non-neovascular eyes.

PURPOSE: Τo evaluate the evolution of macular atrophy (MA) in patients with neovascular AMD (nAMD), compared with their fellow eyes exhibiting dry AMD (dAMD). METHODS: This retrospective study included 124 patients from three centers treated with anti-VEGF in their nAMD eye and having dAMD in the fellow eye. Patients without MA at baseline were analyzed to study the time to first MA development. Synchronous and unsynchronous time course of MA was also studied. MA was evaluated using near-infrared images, while all available optical coherence tomography (OCT) images were used to confirm the criteria proposed by the Classification of Atrophy Meetings group for complete MA. RESULTS: MA first detection in nAMD eyes increased significantly from year 2 to 6 compared to dAMD eyes. Over the study's follow-up, 45.1% of nAMD-E developed MA, compared to 16.5% of fellow eyes (p < 0.001). When MA in the two eyes was compared in a synchronous paired manner over 4 years, nAMD eyes had an average MA progression rate of 0.275 mm/year versus 0.110 mm/year in their fellow dAMD eyes. Multivariate ANOVA revealed significant time (p < 0.001), eye (p = 0.003), and time-eye interaction (p < 0.001) effects. However, when MA did develop in dAMD eyes and was compared in an asynchronous manner to MA of nAMD eyes, it was found to progress faster in dAMD eyes (dAMD: 0.295 mm/year vs. nAMD: 0.176 mm/year) with a significant time-eye interaction (p = 0.015). CONCLUSIONS: In this study, a significant difference in MA incidence and progression was documented in eyes with nAMD under treatment, compared to fellow eye exhibiting dAMD. Eyes with nAMD tended to develop more MA compared to fellow dAMD eyes. However, when atrophy did develop in the fellow dAMD eyes, it progressed faster over time compared to MA in nAMD eyes.

Structure-function association between contrast sensitivity and retinal thickness (total, regional, and individual retinal layer) in patients with idiopathic epiretinal membrane.

PURPOSE: To investigate structure-function associations between retinal thickness, visual acuity (VA), and contrast sensitivity (CS), using the quantitative contrast sensitivity function (qCSF) method in patients with idiopathic epiretinal membrane (ERM). METHODS: Retrospective, cross-sectional observational study. Patients with a diagnosis of idiopathic ERM were included. Patients underwent complete ophthalmic examination, spectral-domain optical coherence tomography imaging (SD-OCT) (SPECTRALIS® Heidelberg), and CS testing using the qCSF method. Outcomes included area under the log CSF (AULCSF), contrast acuity (CA), and CS thresholds at 1, 1.5, 3, 6, 12, and 18 cycles per degree (cpd). RESULTS: A total of 102 eyes of 79 patients were included. Comparing standardized regression coefficients, retinal thickness in most ETDRS sectors was associated with larger reductions in AULCSF, CA, and CS thresholds at 3 and 6 cpd than those in logMAR VA. These differences in effect on VA and CS metrics were more pronounced in the central subfield and inner ETDRS sectors. Among the retinal layers, increased INL thickness had the most detrimental effect on visual function, being significantly associated with reductions in logMAR VA, AULCSF, CA, and CS thresholds at 3 and 6 cpd (all p < .01), as well as at 1.5 and 12 cpd (p < .05). CONCLUSION: Retinal thickness seems to be associated with larger reductions in contrast sensitivity than VA in patients with ERM. Measured with the qCSF method, contrast sensitivity may serve as a valuable adjunct visual function metric for patients with ERM.

COMPLEMENT INHIBITION FOR GEOGRAPHIC ATROPHY: Review of Salient Functional Outcomes and Perspective.

PURPOSE: To evaluate available rationale and outcomes of randomized trial results for complement inhibition for geographic atrophy. METHODS: Data from recently completed randomized trials of complement inhibition, particularly for pegcetacoplan and avacincaptad pegol, were evaluated for both the outcome, area of autofluorescence loss, and functional vision tests. RESULTS: Pegcetacoplan 2 mg showed statistically significant reduction in expansion of the area of autofluorescence loss with monthly, but not every-other-month dosing, in a 12-month phase two trial. Nearly 40% of patients recruited for the monthly arm did not complete the treatment. In two parallel phase 3 studies there was a statistically significant reduction in the area of atrophy in one but not both studies as compared with untreated controls. Data released at 24 months follow-up showed statistically significant reduction in the area of autofluorescence-detected atrophy in both studies compared with sham. Patients did not show functional difference in best-corrected visual acuity, maximum reading speed, Functional Reading Independence Index, and mean microperimetry threshold sensitivities in the treatment versus sham arms. Avacincaptad pegol was evaluated in two randomized pivotal studies and showed a statistically significant reduction in the expansion of autofluorescence loss at 12 months. Patients in the treatment arms did not show any difference as compared with sham in the best-corrected visual acuity or low luminance visual acuity, the only functional outcomes mentioned. Both drugs increased the risk of macular neovascularization. CONCLUSION: Both avacincaptad pegol and pegcetacoplan show significant differences compared with sham in autofluorescence imaging but no benefit in visual function at 12 and 24 months, respectively.

CSF1R inhibition by a small-molecule inhibitor is not microglia specific; affecting hematopoiesis and the function of macrophages.

Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a method for microglia depletion, with the assumption that it does not affect peripheral immune cells. Here, we show that CSF1R inhibition by PLX5622 indeed affects the myeloid and lymphoid compartments, causes long-term changes in bone marrow-derived macrophages by suppressing interleukin 1ß, CD68, and phagocytosis but not CD208, following exposure to endotoxin, and also reduces the population of resident and interstitial macrophages of peritoneum, lung, and liver but not spleen. Thus, small-molecule CSF1R inhibition is not restricted to microglia, causing strong effects on circulating and tissue macrophages that perdure long after cessation of the treatment. Given that peripheral monocytes repopulate the central nervous system after CSF1R inhibition, these changes have practical implications for relevant experimental data.

RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization.

Inflammation plays an important role in pathological angiogenesis. Receptor-interacting protein 1 (RIP1) is highly expressed in inflammatory cells and is known to play an important role in the regulation of apoptosis, necroptosis, and inflammation; however, a comprehensive description of its role in angiogenesis remains elusive. Here, we show that RIP1 is abundantly expressed in infiltrating macrophages during angiogenesis, and genetic or pharmacological inhibition of RIP1 kinase activity using kinase-inactive RIP1K45A/K45A mice or necrostatin-1 attenuates angiogenesis in laser-induced choroidal neovascularization, Matrigel plug angiogenesis, and alkali injury-induced corneal neovascularization in mice. The inhibitory effect on angiogenesis is mediated by caspase activation through a kinase-independent function of RIP1 and RIP3. Mechanistically, infiltrating macrophages are the key target of RIP1 kinase inhibition to attenuate pathological angiogenesis. Inhibition of RIP1 kinase activity is associated with caspase activation in infiltrating macrophages and decreased expression of proangiogenic M2-like markers but not M1-like markers. Similarly, in vitro, catalytic inhibition of RIP1 down-regulates the expression of M2-like markers in interleukin-4-activated bone marrow-derived macrophages, and this effect is blocked by simultaneous caspase inhibition. Collectively, these results demonstrate a nonnecrotic function of RIP1 kinase activity and suggest that RIP1-mediated modulation of macrophage activation may be a therapeutic target of pathological angiogenesis.

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.

Widefield Swept-Source OCT Angiography Metrics Associated with the Development of Diabetic Vitreous Hemorrhage: A Prospective Study.

PURPOSE: To investigate the association among widefield swept-source (SS) OCT angiography (OCTA) metrics and systemic parameters and vitreous hemorrhage (VH) occurrence in eyes with proliferative diabetic retinopathy (PDR). DESIGN: Prospective, observational study. PARTICIPANTS: Fifty-five eyes from 45 adults with PDR, with no history of VH, followed up for at least 3 months. METHODS: All patients underwent widefield SS OCTA (Montage 15 × 15 mm and high-definition (HD)-51 line scan) imaging. Images were evaluated independently by 2 graders for quantitative and qualitative widefield SS OCTA metrics defined a priori. Systemic and ocular parameters and widefield SS OCTA metrics were screened using least absolute shrinkage and selection operator and logistic or Cox regression for variable selection. Firth's bias-reduced logistic regression models (outcome, occurrence of VH) and Cox regression models (outcome, time to occurrence of VH) were used to identify parameters associated with VH occurrence. MAIN OUTCOME MEASURES: Occurrence of VH. RESULTS: Over a median follow-up of 363 days (range, 28-710 days), 13 of 55 PDR eyes (24%) demonstrated VH during the follow-up period. Presence of extensive neovascularizations (odds ratio, 8.05; 95% confidence interval [CI], 1.43-58.56; P = 0.02), defined as neovascularizations with total area of more than 4 disc diameters, and forward neovascularizations (odds ratio, 5.42; 95% CI, 1.26-35.16; P = 0.02) that traversed the posterior hyaloid face into the vitreous were associated with the occurrence of VH. The presence of flat neovascularizations (odds ratio, 0.25; 95% CI, 0.04-1.01; P = 0.05) confined to the posterior hyaloid face was associated with a lower risk of VH with borderline significance. Similarly, presence of extensive neovascularizations (hazard ratio, 18.24; 95% CI, 3.51-119.47; P < 0.001) and forward neovascularizations (hazard ratio, 9.60; 95% CI, 2.07-68.08; P = 0.002) was associated significantly with time to development of VH. CONCLUSIONS: Widefield SS OCTA is useful for evaluating neovascularizations and their relationship with the vitreous. The presence of forward and extensive neovascularizations was associated with the occurrence of VH in patients with PDR. Larger samples and longer follow-up are needed to verify the risk factors and imaging biomarkers for diabetic VH.

Microglia Regulate Neuroglia Remodeling in Various Ocular and Retinal Injuries.

Reactive microglia and infiltrating peripheral monocytes have been implicated in many neurodegenerative diseases of the retina and CNS. However, their specific contribution in retinal degeneration remains unclear. We recently showed that peripheral monocytes that infiltrate the retina after ocular injury in mice become permanently engrafted into the tissue, establishing a proinflammatory phenotype that promotes neurodegeneration. In this study, we show that microglia regulate the process of neuroglia remodeling during ocular injury, and their depletion results in marked upregulation of inflammatory markers, such as Il17f, Tnfsf11, Ccl4, Il1a, Ccr2, Il4, Il5, and Csf2 in the retina, and abnormal engraftment of peripheral CCR2+ CX3CR1+ monocytes into the retina, which is associated with increased retinal ganglion cell loss, retinal nerve fiber layer thinning, and pigmentation onto the retinal surface. Furthermore, we show that other types of ocular injuries, such as penetrating corneal trauma and ocular hypertension also cause similar changes. However, optic nerve crush injury-mediated retinal ganglion cell loss evokes neither peripheral monocyte response in the retina nor pigmentation, although peripheral CX3CR1+ and CCR2+ monocytes infiltrate the optic nerve injury site and remain present for months. Our study suggests that microglia are key regulators of peripheral monocyte infiltration and retinal pigment epithelium migration, and their depletion results in abnormal neuroglia remodeling that exacerbates neuroretinal tissue damage. This mechanism of retinal damage through neuroglia remodeling may be clinically important for the treatment of patients with ocular injuries, including surgical traumas.

A quantitative comparison of four optical coherence tomography angiography devices in healthy eyes.

PURPOSE: Optical coherence tomography angiography (OCT-A) is a novel imaging modality for the diagnosis of chorioretinal diseases. A number of FDA-approved OCT-A devices are currently commercially available, each with unique algorithms and scanning protocols. Although several published studies have compared different combinations of OCT-A machines, there is a lack of agreement on the consistency of measurements across OCT-A devices. Therefore, we conducted a prospective quantitative comparison of four available OCT-A platforms. METHODS: Subjects were scanned on four devices: Optovue RTVue-XR, Heidelberg Spectralis OCT2 module, Zeiss Plex Elite 9000 Swept-Source OCT, and Topcon DRI-OCT Triton Swept-Source OCT. 3 mm × 3 mm images were utilized for analysis. Foveal avascular zone (FAZ) area was separately and independently measured by two investigators. Fractal dimension (FD), superficial capillary plexus (SCP), and deep capillary plexus (DCP) vessel densities (VD) were calculated from binarized images using the Fiji image processing software. SCP and DCP VD were further calculated after images were skeletonized. Repeated measures ANOVA, post hoc tests, and interclass correlation coefficient (ICC) were performed for statistical analysis. RESULTS: Sixteen healthy eyes from sixteen patients were scanned on the four devices. Images of five eyes from the Triton device were excluded due to poor image quality; thus, the authors performed two sets comparisons, one with and one without the Triton machine. FAZ area showed no significant difference across devices with an ICC of > 95%. However, there were statistically significant differences for SCP and DCP VD both before and after skeletonization (p < 0.05). Fractal analysis revealed no significant difference of FD at the SCP; however, a statistically significant difference was found for FD at the DCP layer (p < 0.05). CONCLUSIONS: The results showed that FAZ measurements were consistent across all four devices, while significant differences in VD and FD measurements existed. Therefore, we suggest that for both clinical follow-up and research studies, FAZ area is a useful parameter for OCT-A image analysis when measurements are made on different machines, while VD and FD show significant variability when measured across devices.

BASELINE PREDICTORS ASSOCIATED WITH 3-YEAR CHANGES IN DARK ADAPTATION IN AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To assess the relationship between baseline age-related macular degeneration (AMD) and disease stage, as well as optical coherence tomography features seen in AMD, with 3-year changes in dark adaptation (DA). METHODS: Prospective longitudinal study including patients with AMD and a comparison group (n = 42 eyes, 27 patients). At baseline and 3 years, we obtained color fundus photographs, spectral-domain optical coherence tomography, and rod-mediated DA (20 minutes protocol). Multilevel mixed-effect models were used for analyses, with changes in rod intercept time at 3 years as the primary outcome. As some eyes (n = 11) reached the DA testing ceiling value at baseline, we used 3-year changes in area under the DA curve as an additional outcome. RESULTS: Baseline AMD, AMD stage, and hyperreflective foci on optical coherence tomography were associated with larger changes in rod intercept time at 3 years. When change in area under the DA curve was used as an outcome, in addition to these features, the presence of retinal atrophy and drusenoid pigment epithelial detachment had significant associations. New subretinal drusenoid deposits at 3 years were also associated with more pronounced changes in rod intercept time and area under the DA curve. CONCLUSION: Specific optical coherence tomography features are associated with DA impairments over time, which supports that structural changes predict functional loss over 3 years.

Permanent neuroglial remodeling of the retina following infiltration of CSF1R inhibition-resistant peripheral monocytes.

Previous studies have demonstrated that ocular injury can lead to prompt infiltration of bone-marrow-derived peripheral monocytes into the retina. However, the ability of these cells to integrate into the tissue and become microglia has not been investigated. Here we show that such peripheral monocytes that infiltrate into the retina after ocular injury engraft permanently, migrate to the three distinct microglia strata, and adopt a microglia-like morphology. In the absence of ocular injury, peripheral monocytes that repopulate the retina after depletion with colony-stimulating factor 1 receptor (CSF1R) inhibitor remain sensitive to CSF1R inhibition and can be redepleted. Strikingly, consequent to ocular injury, the engrafted peripheral monocytes are resistant to depletion by CSF1R inhibitor and likely express low CSF1R. Moreover, these engrafted monocytes remain proinflammatory, expressing high levels of MHC-II, IL-1ß, and TNF-α over the long term. The observed permanent neuroglia remodeling after injury constitutes a major immunological change that may contribute to progressive retinal degeneration. These findings may also be relevant to other degenerative conditions of the retina and the central nervous system.

CFH and ARMS2 genetic risk determines progression to neovascular age-related macular degeneration after antioxidant and zinc supplementation.

We evaluated the influence of an antioxidant and zinc nutritional supplement [the Age-Related Eye Disease Study (AREDS) formulation] on delaying or preventing progression to neovascular AMD (NV) in persons with age-related macular degeneration (AMD). AREDS subjects (n = 802) with category 3 or 4 AMD at baseline who had been treated with placebo or the AREDS formulation were evaluated for differences in the risk of progression to NV as a function of complement factor H (CFH) and age-related maculopathy susceptibility 2 (ARMS2) genotype groups. We used published genetic grouping: a two-SNP haplotype risk-calling algorithm to assess CFH, and either the single SNP rs10490924 or 372_815del443ins54 to mark ARMS2 risk. Progression risk was determined using the Cox proportional hazard model. Genetics-treatment interaction on NV risk was assessed using a multiiterative bootstrap validation analysis. We identified strong interaction of genetics with AREDS formulation treatment on the development of NV. Individuals with high CFH and no ARMS2 risk alleles and taking the AREDS formulation had increased progression to NV compared with placebo. Those with low CFH risk and high ARMS2 risk had decreased progression risk. Analysis of CFH and ARMS2 genotype groups from a validation dataset reinforces this conclusion. Bootstrapping analysis confirms the presence of a genetics-treatment interaction and suggests that individual treatment response to the AREDS formulation is largely determined by genetics. The AREDS formulation modifies the risk of progression to NV based on individual genetics. Its use should be based on patient-specific genotype.

Lysosome-associated membrane protein-2 deficiency increases the risk of reactive oxygen species-induced ferroptosis in retinal pigment epithelial cells.

Lysosome-associated membrane protein-2 (LAMP2), is a highly glycosylated lysosomal membrane protein involved in chaperone mediated autophagy. Mutations of LAMP2 cause the classic triad of myopathy, cardiomyopathy and encephalopathy of Danon disease (DD). Additionally, retinopathy has also been observed in young DD patients, leading to vision loss. Emerging evidence show LAMP2-deficiency to be involved in oxidative stress (ROS) but the mechanism remains obscure. In the present study, we found that tert-butyl hydroperoxide or antimycin A induced more cell death in LAMP2 knockdown (LAMP2-KD) than in control ARPE-19 cells. Mechanistically, LAMP2-KD reduced the concentration of cytosolic cysteine, resulting in low glutathione (GSH), inferior antioxidant capability and mitochondrial lipid peroxidation. ROS induced RPE cell death through ferroptosis. Inhibition of glutathione peroxidase 4 (GPx4) increased lethality in LAMP2-KD cells compared to controls. Cysteine and glutamine supplementation restored GSH and prevented ROS-induced cell death of LAMP2-KD RPE cells.

Biallelic RP1-associated retinal dystrophies: Expanding the mutational and clinical spectrum.

Purpose: To evaluate the phenotypic spectrum of autosomal recessive RP1-associated retinal dystrophies and assess genotypic associations. Methods: A retrospective multicenter study was performed of patients with biallelic RP1-associated retinal dystrophies. Data including presenting symptoms and age, visual acuity, kinetic perimetry, full field electroretinogram, fundus examination, multimodal retinal imaging, and RP1 genotype were evaluated. Results: Nineteen eligible patients from 17 families were identified and ranged in age from 10 to 56 years at the most recent evaluation. Ten of the 21 unique RP1 variants identified were novel, and mutations within exon 2 accounted for nearly half of alleles across the cohort. Patients had clinical diagnoses of retinitis pigmentosa (13), cone-rod dystrophy (3), Leber congenital amaurosis (1), early-onset severe retinal dystrophy (1), and macular dystrophy (1). Macular atrophy was a common feature across the cohort. Symptom onset occurred between 4 and 30 years of age (mean 14.9 years, median 13 years), but there were clusters of onset age that correlated with the effects of RP1 mutations at a protein level. Patients with later-onset disease, including retinitis pigmentosa, had at least one missense variant in an exon 2 DCX domain. Conclusions: Biallelic RP1 mutations cause a broad spectrum of retinal disease. Exon 2 missense mutations are a significant contributor to disease and can be associated with a considerably later onset of retinitis pigmentosa than that typically associated with biallelic RP1 mutations.

Urine Nuclear Magnetic Resonance (NMR) Metabolomics in Age-Related Macular Degeneration.

Biofluid biomarkers of age-related macular degeneration (AMD) are still lacking, and their identification is challenging. Metabolomics is well-suited to address this need, and urine is a valuable accessible biofluid. This study aimed to characterize the urinary metabolomic signatures of patients with different stages of AMD and a control group (>50 years). It was a prospective, cross-sectional study, where subjects from two cohorts were included: 305 from Coimbra, Portugal (AMD patients n = 252; controls n = 53) and 194 from Boston, United States (AMD patients n = 147; controls n = 47). For all participants, we obtained color fundus photographs (for AMD staging) and fasting urine samples, which were analyzed using 1H nuclear magnetic resonance (NMR) spectroscopy. Our results revealed that in both cohorts, urinary metabolomic profiles differed mostly between controls and late AMD patients, but important differences were also found between controls and subjects with early AMD. Analysis of the metabolites responsible for these separations revealed that, even though distinct features were observed for each cohort, AMD was in general associated with depletion of excreted citrate and selected amino acids at some stage of the disease, suggesting enhanced energy requirements. In conclusion, NMR metabolomics enabled the identification of urinary signals of AMD and its severity stages, which might represent potential metabolomic biomarkers of the disease.