Mouse Model Of Age-related Macular Degeneration Research Articles

Retinoic acid related orphan receptor α is a genetic modifier that rescues retinal degeneration in a mouse model of Stargardt disease and Dry AMD.

Degeneration of the macula is associated with several overlapping diseases including age-related macular degeneration (AMD) and Stargardt Disease (STGD). Mutations in ATP Binding Cassette Subfamily A Member 4 (ABCA4) are associated with late-onset dry AMD and early-onset STGD. Additionally, both forms of macular degeneration exhibit deposition of subretinal material and photoreceptor degeneration. Retinoic acid related orphan receptor α (RORA) regulates the AMD inflammation pathway that includes ABCA4, CD59, C3 and C5. In this translational study, we examined the efficacy of RORA at attenuating retinal degeneration and improving the inflammatory response in Abca4 knockout (Abca4-/-) mice. AAV5-hRORA-treated mice showed reduced deposits, restored CD59 expression and attenuated amyloid precursor protein (APP) expression compared with untreated eyes. This molecular rescue correlated with statistically significant improvement in photoreceptor function. This is the first study evaluating the impact of RORA modifier gene therapy on rescuing retinal degeneration. Our studies demonstrate efficacy of RORA in improving STGD and dry AMD-like disease.

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

Targeting ZIP8 mediated ferroptosis as a novel strategy to protect against the retinal pigment epithelial degeneration

The degeneration of retinal pigment epithelium (RPE) plays an important role in the development of age-related macular degeneration (AMD). However, the underlying mechanism remains elusive. In this study, we identified that ZIP8, a metal-ion transporter, plays a crucial role in the degeneration of RPE cells mediated by ferroptosis. ZIP8 was found to be upregulated in patients with AMD through transcriptome analysis. Upregulated ZIP8 was also observed in both oxidative-stressed RPE cells and AMD mouse model. Importantly, knockdown of ZIP8 significantly inhibited ferroptosis in RPE cells induced by sodium iodate-induced oxidative stress. Blocking ZIP8 with specific antibodies reversed RPE degeneration and restored retinal function, improving visual loss in a mouse model of NaIO3-induced. Interestingly, the modification of the N-glycosylation sites N40, N72 and N88, but not N273, was essential for the intracellular iron accumulation mediated by ZIP8, which further led to increased lipid peroxidation and RPE death. These findings highlight the critical role of ZIP8 in RPE ferroptosis and provide a potential target for the treatment of diseases associated with retinal degeneration, including AMD.

Microglia at sites of atrophy restrict the progression of retinal degeneration via galectin-3 and Trem2.

Outer retinal degenerations, including age-related macular degeneration (AMD), are characterized by photoreceptor and retinal pigment epithelium (RPE) atrophy. In these blinding diseases, macrophages accumulate at atrophic sites, but their ontogeny and niche specialization remain poorly understood, especially in humans. We uncovered a unique profile of microglia, marked by galectin-3 upregulation, at atrophic sites in mouse models of retinal degeneration and human AMD. In disease models, conditional deletion of galectin-3 in microglia led to phagocytosis defects and consequent augmented photoreceptor death, RPE damage, and vision loss, indicating protective roles. Mechanistically, Trem2 signaling orchestrated microglial migration to atrophic sites and induced galectin-3 expression. Moreover, pharmacologic Trem2 agonization led to heightened protection but in a galectin-3-dependent manner. In elderly human subjects, we identified this highly conserved microglial population that expressed galectin-3 and Trem2. This population was significantly enriched in the macular RPE-choroid of AMD subjects. Collectively, our findings reveal a neuroprotective population of microglia and a potential therapeutic target for mitigating retinal degeneration.

Fluorescence Lifetime Imaging Ophthalmoscopy of Mouse Models of Age-related Macular Degeneration.

To investigate fluorescence lifetime of mouse models of age-related macular degeneration (AMD) by fluorescence lifetime imaging ophthalmoscopy (FLIO). Two AMD mouse models, apolipoprotein E knockout (ApoE-/-) mice and NF-E2-related factor-2 knockout (Nrf2-/-) mice, and their wild-type mice underwent monthly ophthalmic examinations including FLIO from 3months of age. After euthanasia at the age of 6 or 11months, blood plasma was collected to determine total antioxidant capacity and eyes were enucleated for Oil red O (ORO) lipid staining of chorioretinal tissue. In FLIO, the mean fluorescence lifetime (τm) of wild type shortened with age in both spectral channels. In short spectral channel, τm shortening was observed in both AMD models as well, but its rate was more pronounced in ApoE-/- mice and significantly different from the other strains as months of age progressed. In contrast, in long spectral channel, both model strains showed completely opposite trends, with τm becoming shorter in ApoE-/- and longer in Nrf2-/- mice than the others. Oil red O staining at Bruch's membrane was significantly stronger in ApoE-/- mice at 11months than the other strains. Plasma total antioxidant capacity was highest in ApoE-/- mice at both 6 and 11months. The two AMD mouse models exhibited largely different fundus fluorescence lifetime, which might be related to the different systemic metabolic state. FLIO might be able to indicate different metabolic states of eyes at risk for AMD. This animal study may provide new insights into the relationship between early AMD-associated metabolic changes and FLIO findings.

Age-Related Changes in the Glycolytic Enzymes of M2-Isoform of Pyruvate Kinase and Fructose-1,6-Bisphosphate Aldolase: Implications to Age-Related Macular Degeneration.

Prior studies have emphasized a bioenergetic crisis in the retinal pigment epithelium (RPE) as a critical factor in the development of age-related macular degeneration (AMD). The isoforms Fructose-1,6-bisphosphate aldolase C (ALDOC) and pyruvate kinase M2 (PKM2) have been proposed to play a role in AMD pathogenesis. While PKM2 and ALDOC are crucial for aerobic glycolysis in the neural retina, they are not as essential for the RPE. In this study, we examined the expression and activity of PKM2 and ALDOC in both young and aged RPE cells, as well as in the retina and RPE tissue of mice, including an experimentally induced AMD mouse model. Our findings reveal an upregulation in PKM2 and ALDOC expression, accompanied by increased pyruvate kinase activity, in the aged and AMD mouse RPE. Conversely, there is a decrease in ALDOC expression but an increase in PKM2 expression and pyruvate kinase activity in the aged and AMD retina. Overall, our study indicates that aged and AMD RPE cells tend to favor aerobic glycolysis, while this tendency is diminished in the aged and AMD retina. These results underscore the significance of targeting PKM2 and ALDOC in the RPE as a promising therapeutic approach to address the bioenergetic crisis and prevent vision loss in AMD.

Mesoporous Cerium Oxide Nanoparticles with High Scavenging Properties of Reactive Oxygen Species for Treating Age‐Related Macular Degeneration

Age‐related macular degeneration (AMD), the leading cause of vision loss among older individuals, is characterized by damage to photoreceptors and retinal pigment epithelial cells (RPEs). Oxidative stress and chronic inflammation in the retina play notable roles in AMD pathogenesis, rendering them attractive therapeutic targets. Cerium oxide nanoparticles (CeNPs) have shown promise in scavenging reactive oxygen species (ROS) by mimicking antioxidant enzymes, whereas mesoporous materials have emerged as versatile drug carriers. Herein, mesoporous CeNPs (mCeNPs) that integrate the advantages of CeNPs and mesoporous materials are presented. The mCeNPs can be synthesized using 1,1′‐carbonyldiimidazole and imidazole in acetone without heating and pressurization. The resulting mCeNPs exhibit mesoporous structures comprising assembled small CeNPs, exerting excellent ROS‐scavenging capabilities, biocompatibility, and cytoprotective and anti‐inflammatory effects against H2O2‐induced damage in RPEs. Using a sodium iodate‐induced AMD mouse model, it is demonstrated that intravitreal mCeNP administration can exhibit disease‐preventive effects. These findings indicate the therapeutic potential of mCeNPs against AMD.

Thrombospondin-1 proteomimetic polymers exhibit anti-angiogenic activity in a neovascular age-related macular degeneration mouse model.

Neovascular age-related macular degeneration (nAMD) is the leading cause of blindness in the developed world. Current therapy includes monthly intraocular injections of anti-VEGF antibodies, which are ineffective in up to one third of patients. Thrombospondin-1 (TSP1) inhibits angiogenesis via CD36 binding, and its down-regulated expression is negatively associated with the onset of nAMD. Here, we describe TSP1 mimetic protein-like polymers (TSP1 PLPs). TSP1 PLPs bind CD36 with high affinity, resist degradation, show prolonged intraocular half-lives (13.1 hours), have no toxicity at relevant concentrations in vivo (40 μM), and are more efficacious in ex vivo choroidal sprouting assays compared to the peptide sequence and Eylea (aflibercept), the current standard of care anti-VEGF treatment. Furthermore, PLPs exhibit superior in vivo efficacy in a mouse model for nAMD compared to control PLPs consisting of scrambled peptide sequences, using fluorescein angiography and immunofluorescence. Since TSP-1 inhibits angiogenesis by VEGF-dependent and independent mechanisms, TSP1 PLPs are a potential therapeutic for patients with anti-VEGF treatment-resistant nAMD.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Supramolecular Senolytics via Intracellular Oligomerization of Peptides in Response to Elevated Reactive Oxygen Species Levels in Aging Cells.

Senolytics, which eliminate senescent cells from tissues, represent an emerging therapeutic strategy for various age-related diseases. Most senolytics target antiapoptotic proteins, which are overexpressed in senescent cells, limiting specificity and inducing severe side effects. To overcome these limitations, we constructed self-assembling senolytics targeting senescent cells with an intracellular oligomerization system. Intracellular aryl-dithiol-containing peptide oligomerization occurred only inside the mitochondria of senescent cells due to selective localization of the peptides by RGD-mediated cellular uptake into integrin αvβ3-overexpressed senescent cells and elevated levels of reactive oxygen species, which can be used as a chemical fuel for disulfide formation. This oligomerization results in an artificial protein-like nanoassembly with a stable α-helix secondary structure, which can disrupt the mitochondrial membrane via multivalent interactions because the mitochondrial membrane of senescent cells has weaker integrity than that of normal cells. These three specificities (integrin αvβ3, high ROS, and weak mitochondrial membrane integrity) of senescent cells work in combination; therefore, this intramitochondrial oligomerization system can selectively induce apoptosis of senescent cells without side effects on normal cells. Significant reductions in key senescence markers and amelioration of retinal degeneration were observed after elimination of the senescent retinal pigment epithelium by this peptide senolytic in an age-related macular degeneration mouse model and in aged mice, and this effect was accompanied by improved visual function. This system provides a strategy for the treatment of age-related diseases using supramolecular senolytics.

A Protocol to Evaluate and Quantify Retinal Pigmented Epithelium Pathologies in Mouse Models of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a debilitating retinal disorder in aging populations. It is widely believed that dysfunction of the retinal pigmented epithelium (RPE) is a key pathobiological event in AMD. To understand the mechanisms that lead to RPE dysfunction, mouse models can be utilized by researchers. It has been established by previous studies that mice can develop RPE pathologies, some of which are observed in the eyes of individuals diagnosed with AMD. Here, we describe a phenotyping protocol to assess RPE pathologies in mice. This protocol includes the preparation and evaluation of retinal cross-sections using light microscopy and transmission electron microscopy, as well as that of RPE flat mounts by confocal microscopy. We detail the common types of murine RPE pathologies observed by these techniques and ways to quantify them through unbiased methods for statistical testing. As proof of concept, we use this RPE phenotyping protocol to quantify the RPE pathologies observed in mice overexpressing transmembrane protein 135 (Tmem135) and aged wild-type C57BL/6J mice. The main goal of this protocol is to present standard RPE phenotyping methods with unbiased quantitative assessments for scientists using mouse models of AMD.

Repeated Topical Administration of 3 nm Cerium Oxide Nanoparticles Reverts Disease Atrophic Phenotype and Arrests Neovascular Degeneration in AMD Mouse Models.

Increased oxidative stress in the retina and retinal pigment epithelium is implicated in age-related macular degeneration (AMD). Antioxidant cerium oxide nanoparticles (CeO2NPs) have been used to treat degenerative retinal pathologies in animal models, although their delivery route is not ideal for chronic patient treatment. In this work, we prepared a formulation for ocular topical delivery that contains small (3 nm), nonaggregated biocompatible CeO2NPs. In vitro results indicate the biocompatible and protective character of the CeO2NPs, reducing oxidative stress in ARPE19 cells and inhibiting neovascularization related to pathological angiogenesis in both HUVEC and in in vitro models of neovascular growth. In the in vivo experiments, we observed the capacity of CeO2NPs to reach the retina after topical delivery and a subsequent reversion of the altered retinal transcriptome of the retinal degenerative mouse model DKOrd8 toward that of healthy control mice, together with signs of decreased inflammation and arrest of degeneration. Furthermore, CeO2NP eye drops' treatment reduced laser-induced choroidal neovascular lesions in mice by lowering VEGF and increasing PEDF levels. These results indicate that CeO2NP eye drops are a beneficial antioxidant and neuroprotective treatment for both dry and wet forms of AMD disease.

Retinal protection by fungal product theissenolactone B in a sodium iodate-induced AMD model through targeting retinal pigment epithelial matrix metalloproteinase-9 and microglia activity

Age-related macular degeneration (AMD) is the leading cause of low vision and blindness for which there is currently no cure. Increased matrix metalloproteinase-9 (MMP-9) was found in AMD and potently contributes to its pathogenesis. Resident microglia also promote the processes of chronic neuroinflammation, accelerating the progression of AMD. The present study investigates the effects and mechanisms of the natural compound theissenolactone B (LB53), isolated from Theissenia cinerea, on the effects of RPE dysregulation and microglia hyperactivation and its retinal protective ability in a sodium iodate (NaIO3)-induced retinal degeneration model of AMD. The fungal component LB53 significantly reduces MMP-9 gelatinolysis in TNF-α-stimulated human RPE cells (ARPE-19). Similarly, LB53 abolishes MMP-9 protein and mRNA expression in ARPE-19 cells. Moreover, LB53 efficiently suppresses nitric oxide (NO) production, iNOS expression, and intracellular ROS levels in LPS-stimulated TLR 4-activated microglial BV-2 cells. According to signaling studies, LB53 specifically targets canonical NF-κB signaling in both ARPE-19 and BV-2 microglia. In an RPE-BV-2 interaction assay, LB53 ameliorates LPS-activated BV-2 conditioned medium-induced MMP-9 activation and expression in the RPE. In NaIO3-induced AMD mouse model, LB53 restores photoreceptor and bipolar cell dysfunction as assessed by electroretinography (ERG). Additionally, LB53 prevents retinal thinning, primarily the photoreceptor, and reduces retinal blood flow from NaIO3 damage evaluated by optic coherence tomography (OCT) and laser speckle flowgraphy (LSFG), respectively. Our results demonstrate that LB53 exerts neuroprotection in a mouse model of AMD, which can be attributed to its anti-retinal inflammatory effects by impeding RPE-mediated MMP-9 activation and anti-microglia.

PGE2 promotes macrophage recruitment and neovascularization in murine wet-type AMD models

Age-related macular degeneration (AMD), a progressive chronic disease of the central retina, is a leading cause of blindness worldwide. Activated macrophages recruited to the injured eyes greatly contribute to the pathogenesis of choroidal neovascularization (CNV) in exudative AMD (wet AMD). This study describes the effects of cyclooxygenase-2 (COX2)/prostaglandin E2 (PGE2) signalling on the macrophage activation and CNV formation of wet AMD. In a mouse model of laser-induced wet AMD, the mice received an intravitreal injection of celecoxib (a selective COX2 inhibitor). Optical coherence tomography (OCT), fundus fluorescein angiography (FFA), choroidal histology of the CNV lesions, and biochemical markers were assessed. The level of PGE2 expression was high in the laser-induced CNV lesions. Macrophage recruitment and CNV development were significantly less after celecoxib treatment. E-prostanoid1 receptor (EP1R)/protein kinase C (PKC) signalling was involved in M2 macrophage activation and interleukin-10 (IL-10) production of bone marrow-derived macrophages (BMDMs) in vitro. In addition, IL-10 was found to induce the proliferation and migration of human choroidal microvascular endothelial cells (HCECs). Thus, the PGE2/EP1R signalling network serves as a potential therapeutic target for CNV of the wet-type AMD.Graphical AXv86vuL_n_YsWmKFp4z_2Video abstract

Increased efficacy of dietary supplement containing wax ester-rich marine oil and xanthophylls in a mouse model of dry macular degeneration.

Age-related macular degeneration (AMD) is nowadays considered among the retinal diseases whose clinical management lacks established treatment approaches, mainly for its atrophic (dry) form. In this respect, the use of dietary patterns enriched in omega-3 and antioxidant xanthophylls has emerged as a promising approach to counteract dry AMD progression although the prophylactic potential of omega-3 of fish origin has been discussed. Whether enriched availability of omega-3 and xanthophylls may increase the effectiveness of diet supplementation in preventing dry AMD remains to be fully established. The present study aims at comparing the efficacy of an existing orally administered formulation based on lutein and fish oil, as a source of omega-3, with a novel formulation providing the combination of lutein and astaxanthin with Calanus oil (COil), which contains omega-3 together with their precursors policosanols. Using a mouse model of dry AMD based on subretinal injection of polyethylene glycol (PEG)-400, we assessed the comparative efficacy of both formulations on PEG-induced major hallmarks including oxidative stress, inflammation, glial reactivity and outer retinal thickness. Dietary supplementation with both mixtures has been found to exert a significant antioxidant and anti-inflammatory activity as reflected by the overall amelioration of the PEG-induced pathological hallmarks. Noteworthy, the formulation based on COil appeared to be more protective than the one based on fish oil, presumably because of the higher bioavailability of omega-3 in COil. These results support the use of dietary supplements combining omega-3 and xanthophylls in the prevention and treatment of AMD and suggest that the source of omega-3 might contribute to treatment efficacy.

Potential gene identification and pathway crosstalk analysis of age-related macular degeneration.

Age-related macular degeneration (AMD), the most prevalent visual disorder among the elderly, is confirmed as a multifactorial disease. Studies demonstrated that genetic factors play an essential role in its pathogenesis. Our study aimed to make a relatively comprehensive study about biological functions of AMD related genes and crosstalk of their enriched pathways. 1691 AMD genetic studies were reviewed, GO enrichment and pathway crosstalk analyses were conducted to elucidate the biological features of these genes and to demonstrate the pathways that these genes participate. Moreover, we identified novel AMD-specific genes using shortest path algorithm in the context of human interactome. We retrieved 176 significantly AMD-related genes. GO results showed that the most significant term in each of these three GO categories was: signaling receptor binding (PBH = 4.835 × 10−7), response to oxygen-containing compound (PBH = 2.764 × 10−21), and extracellular space (PBH = 2.081 × 10−19). The pathway enrichment analysis showed that complement pathway is the most enriched. The pathway crosstalk study showed that the pathways could be divided into two main modules. These two modules were connected by cytokine-cytokine receptor interaction pathway. 42 unique genes potentially participating AMD development were obtained. The aberrant expression of the mRNA of FASN and LRP1 were validated in AMD cell and mouse models. Collectively, our study carried out a comprehensive analysis based on genetic association study of AMD and put forward several evidence-based genes for future study of AMD.

P2X7-mediated alteration of membrane fluidity is associated with the late stages of age-related macular degeneration.

We have shown deficits in monocyte phagocytosis from patients with age-related macular degeneration (AMD). Cell membrane fluidity is known to affect phagocytic capacity and leucocyte functionality more generally. Therefore, we examined membrane fluidity of peripheral blood leucocytes in human patients with AMD and in the P2X7 null mouse model of AMD using flow cytometry with a fluorescent probe for fluidity, TMA-DPH. The results showed that membrane fluidity was decreased in all leucocyte types of late AMD relative to healthy controls (HC) including monocytes, neutrophils and lymphocytes but this was not apparent in earlier stages of AMD. Further analysis of factors contributing to membrane fluidity indicated that pre-treatment of monocytes and lymphocytes with ATP greatly increased membrane fluidity in humans and mice. Evidence from P2X7 null mice and P2X7 antagonists confirmed that these ATP-driven increases in membrane fluidity were mediated by P2X7 but were not associated with the classic P2X7 functions of pore formation or phagocytosis. Analysis of P2X7 expression indicated that receptor levels were elevated in classic monocytes of late AMD patients, further suggesting the P2X7 may contribute to altered plasma membrane properties. Our findings identified a novel biological function of P2X7 in modulating membrane fluidity of leucocytes and demonstrated reduced membrane fluidity in cellular changes associated with the late stage of AMD.

Downregulation of Inflammatory Response via Nrf2/Trx1/TXNIP Axis in Oxidative Stress-Induced ARPE-19 Cells and Mouse Model of AMD.

Aim Chronic inflammation is crucial for age-related macular degeneration (AMD) pathogenesis. However, the mechanism involved in activating inflammation remains unclear. This study is aimed at investigating whether nuclear factor erythrocyte-associated factor 2 (Nrf2) negatively regulated the Nod-like receptor protein 3 (NLRP3) inflammasomes through the thioredoxin 1 (Trx1)/thioredoxin interaction protein (TXNIP) complex. Methods We determined the optimal hydrogen peroxide (H2O2) concentration, time, and changes in reactive oxygen species (ROS) levels. We also constructed animal models using blue LED irradiation. Then, the expression of Nrf2, TXNIP, Trx1, NLRP3, and inflammation-related factors and proteins, along with the changes in retinal thickness and functional status, was analyzed. Results The oxidative stress model was established after 1 h intervention with 100 μM H2O2. Nrf2 reduced ROS production, protected the ultrastructure of mitochondria, increased the thickness of the ONL layer, and increased the amplitude of a- and b-wave amplitudes in ERG. Trx1 knockdown increased the production of ROS, damaged the ultrastructure of mitochondria, reduced the thickness of the other ONL layer, and reduced the amplitudes of a- and b-waves in the electroretinogram (ERG). Thus, TXNIP in the cytoplasm activated the inflammasomes. Conclusions Nrf2 showed antioxidant and anti-inflammatory activity in the H2O2-induced cell stress model and blue LED-induced retinal light damage model. TXNIP transferred from the nucleus to the cytoplasm, activated NLRP3, and aggravated the retinal injury in both the cell stress model and the animal blue LED model. In contrast, Trx1 knockout promoted this process. This study revealed the possible role of the thioredoxin system in developing AMD while also providing newer insights for the future treatment of AMD.

Increased LCN2 (lipocalin 2) in the RPE decreases autophagy and activates inflammasome-ferroptosis processes in a mouse model of dry AMD

ABSTRACT In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options. Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1β: interleukin 1 beta; IS: Inner segment; ITGB1/integrin β1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.

Fibroblast growth factor-21 alleviates phenotypic characteristics of dry age-related macular degeneration in mice

Age-related macular degeneration (AMD) is the main cause of blindness in elderly individuals. As a metabolic regulator, fibroblast growth factor 21 (FGF-21) has been proven indicated to have an effect on wet AMD, but whether this cytokine has a therapeutic effect on dry AMD is unclear. The current study aimed to evaluate the preventive effects of FGF-21 against retinal degeneration in mice and provide mechanistic insights. FGF-21−/− mice were raised to 10 months of age. Then, the morphological changes in the retinal pigment epithelium (RPE)/choroid of the mice were observed by transmission electron microscopy (TEM), and iTRAQ was used to detect the variations in the protein profile. Next, FGF-21−/− and wild-type mice of the same age were fed hydroquinone to generate a dry AMD mouse model to examine whether exogenous FGF-21 can interfere with the occurrence and development of dry AMD. In vivo studies revealed that following FGF-21 knockout, there was an increase in the expression of complement in the RPE/choroid concomitant with the occurrence of dry AMD-like pathological changes. Furthermore, exogenous FGF-21 administration effectively reversed this phenomenon. FGF-21 also demonstrated strong anti-inflammatory effects in the RPE/choroid by inhibiting the NF-κB pathway. In conclusion, the present study demonstrates that FGF-21 treatment presents a novel therapeutic approach for the prevention and development of dry AMD by reducing complement.