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Matrix metalloproteinase-9 (MMP9) and total amyloid-beta (Aβ) are prospective biomarkers of ocular ageing and retinopathy. These were quantified by ELISA in the vitreous and blood from controls (n = 55) and in a subset of age-related macular degeneration (AMD) patients (n = 12) for insights and possible additional links between the ocular and systemic compartments. Vitreous MMP9 levels in control and AMD groups were 932.5 ± 240.9 pg/mL and 813.7 ± 157.6 pg/mL, whilst serum levels were 2228 ± 193 pg/mL and 2386.8 ± 449.4 pg/mL, respectively. Vitreous Aβ in control and AMD groups were 1173.5 ± 117.1 pg/mL and 1275.6 ± 332.9 pg/mL, whilst plasma Aβ were 574.3 ± 104.8 pg/mL and 542.2 ± 139.9 pg/mL, respectively. MMP9 and Aβ showed variable levels across the lifecourse, indicating no correlation to each other or with age nor AMD status, though the smaller AMD cohort was a limiting factor. Aβ and MMP9 levels in the vitreous and blood were unrelated to mean arterial pressure. Smoking, another modifiable risk, showed no association with vitreous Aβ. However, smoking may be linked with vitreous (p = 0.004) and serum (p = 0.005) MMP9 levels in control and AMD groups, though this did not reach our elevated (p = 0.001) significance. A bioinformatics analysis revealed promising MMP9 and APP/Aβ partners for further scrutiny, many of which are already linked with retinopathy.

Scope: The intake of a “Western-style” diet rich in fats is linked with developing retinopathies including age-related macular degeneration (AMD). Wildtype mice are given a high fat diet (HFD) to determine how unhealthy foods can bring about retinal degeneration. Methods and results: Following weaning, female C57BL/6 mice are maintained on standard chow (7% kcal fat, n = 29) or a HFD (45% kcal fat, n = 27) for 12 months. Animals were sacrificed following electroretinography (ERG) and their eyes analyzed by histology, confocal immunofluorescence, and transmission electron microscopy. HFD mice become obese, but showed normal retinal function compared to chow-fed controls. However, diminished β3tubulin labeling of retinal cross-sections indicated fewer/damaged neuronal processes in the inner plexiform layer. AMD-linked proteins clusterin and TIMP3 accumulated in the retinal pigment epithelium (RPE) and Bruch's membrane (BrM). Neutral lipids also deposited in the outer retinae of HFD mice. Ultrastructural analysis revealed disorganized photoreceptor outer segments, collapsed/misaligned RPE microvilli, vacuoles, convoluted basolateral RPE infolds and BrM changes. Basal laminar-like deposits were also present alongside abnormal choroidal endothelial cells. Conclusions: We show that prolonged exposure to an unhealthy “Western-style” diet alone can recapitulate early-intermediate AMD-like features in wildtype mice, highlighting the importance of diet and nutrition in the etiology of sight-loss.

Sorsby fundus dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease-related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting that mutant TIMP3 dimerisation does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of ECM proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-hiPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, PDGF and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis-related changes in SFD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nystagmus is a disorder of uncontrolled eye movement and can occur as an isolated trait (idiopathic INS, IINS) or as part of multisystem disorders such as albinism, significant visual disorders or neurological disease. Eighty-one unrelated patients with nystagmus underwent routine ocular phenotyping using commonly available phenotyping methods and were grouped into four sub-cohorts according to the level of phenotyping information gained and their findings. DNA was extracted and sequenced using a broad utility next generation sequencing (NGS) gene panel. A clinical subpanel of genes for nystagmus/albinism was utilised and likely causal variants were prioritised according to methods currently employed by clinical diagnostic laboratories. We determine the likely underlying genetic cause for 43.2% of participants with similar yields regardless of prior phenotyping. This study demonstrates that a diagnostic workflow combining basic ocular phenotyping and a clinically available targeted NGS panel, can provide a high diagnostic yield for patients with infantile nystagmus, enabling access to disease specific management at a young age and reducing the need for multiple costly, often invasive tests. By describing diagnostic yield for groups of patients with incomplete phenotyping data, it also permits the subsequent design of ‘real-world’ diagnostic workflows and illustrates the changing role of genetic testing in modern diagnostic workflows for heterogeneous ophthalmic disorders.

Alzheimer’s disease-associated amyloid beta (Aβ) proteins accumulate in the outer retina with increasing age and in eyes of age-related macular degeneration (AMD) patients. To study Aβ-induced retinopathy, wild-type mice were injected with nanomolar human oligomeric Aβ1-42, which recapitulate the Aβ burden reported in human donor eyes. In vitro studies investigated the cellular effects of Aβ in endothelial and retinal pigment epithelial (RPE) cells. Results show subretinal Aβ-induced focal AMD-like pathology within 2 weeks. Aβ exposure caused endothelial cell migration, and morphological and barrier alterations to the RPE. Aβ co-localized to late-endocytic compartments of RPE cells, which persisted despite attempts to clear it through upregulation of lysosomal cathepsin B, revealing a novel mechanism of lysosomal impairment in retinal degeneration. The rapid upregulation of cathepsin B was out of step with the prolonged accumulation of Aβ within lysosomes, and contrasted with enzymatic responses to internalized photoreceptor outer segments (POS). Furthermore, RPE cells exposed to Aβ were identified as deficient in cargo-carrying lysosomes at time points that are critical to POS degradation. These findings imply that Aβ accumulation within late-endocytic compartments, as well as lysosomal deficiency, impairs RPE function over time, contributing to visual defects seen in aging and AMD eyes.

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy. We used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central mouse retina. Three-dimensional-reconstructed OS revealed the RPE to support large numbers of photoreceptors (90–216 per RPE cell). Larger bi-nucleate RPE maintained more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE supporting fewer photoreceptors. Scrutiny of RPE microvilli and interdigitating OS revealed the angle and surface area of contact between RPE and photoreceptors. Bi-nucleate RPE contained more mitochondria compared to mono-nucleate RPE. Furthermore, bi-nucleate cells contained larger sub-RPE spaces, supporting a likely association with disease. Use of perfusion-fixed tissues ensured the highest possible standard of preservation, providing novel insights into the 3D RPE architecture and changes linked with retinopathy. This study serves as a benchmark for comparing retinal tissues from donor eyes with age-related macular degeneration (AMD) and other retinopathies.

Advances in imaging have led to the development of several new types of microscopes such as serial block face scanning electron microscopy (SBF-SEM), lightsheet microscopy, as well as X-ray micro-computed tomography (micro-CT), which enables the study of samples in fundamentally different ways. Significantly, these are now commercially available, which facilitates their widespread use in research. With SBF-SEM, fixed and resin-embedded specimens can be serially sectioned and imaged to construct a 3D dataset of the ultrastructure of cells and tissues at high resolution. We used this technique on perfusion-fixed C57BL/6 mouse eyes to image the outer retina. Our findings revealed novel organizational details of the retinal pigment epithelium (RPE) (Keeling et al., 2020b); a specialized cell monolayer that maintains the overlying photoreceptors and also forms the outer blood-retinal-barrier. RPE cells were found to look after far more photoreceptors than was widely assumed. 3D-data enabled measurements of the RPE cytoplasmic and nuclear volumes, the length and angle of microvilli on the apical RPE surface, as well as sub-RPE spaces under the basolateral membrane. The study also compared between mono-nucleate vs. bi-nucleate RPE cells, whilst the use of computing microinstructions (macros) provided information on interactions between adjacent cells in the RPE monolayer. Analysis of SBF-SEM stacks showed several hundred mitochondria which were rendered in 3D, providing information on their volume and spatial distribution in healthy RPE. Mitochondria were found in varying shapes and sizes, and predominantly localized to the mid and basal-zones of cells. The capabilities of SBF-SEM alongside other imaging techniques are being increasingly harnessed by investigators to gain novel insights into the organization of cells and tissues in the eye. These findings also help improve the current understanding of pathology linked with common blinding conditions such as age-related macular degeneration (AMD), as well as rare forms of retinopathy which leads to irreversible sight-loss

Loss of central vision critical to everyday activities such as reading, face-recognition and driving due to damage in the central retina (the macula) is the leading cause of irreversible blindness amongst adults in the developed world. This condition, termed age-related macular degeneration (AMD), is a complex, chronic degenerative disease driven by a combination of genetic and lifestyle risk factors. Early signs of retinal changes in people as young as 30-40 years have been reported, although these individuals appear to be asymptomatic. However, by the age of 65 the disease is present in ~3% of individuals, which increases dramatically to affect 1/3 of individuals by the eighth decade of life. Early to intermediate AMD is estimated to affect ~150 million individuals globally, with another 10 million individuals suffering from end-stage, sight-threatening forms. These terminal stages are broadly grouped into dry (GA: geographic atrophy) or wet (CNV: choroidal neovascular) AMD (Sarks et al., 1988; Bird et al., 2014), with similar frequencies reported in patients. Recent advances in identifying genetic risk factors, including our discoveries in this field, indicate an initial shared pathology before progressing to aforementioned late-stage phenotypes. Currently, GA patients have no effective treatment, which may in part be due to the lack of good in-vivo models for GA studies. Here, we summarise our new findings that describe an altogether new mouse model with GA-like features which shows progressive outer retinal pathology (Ibbett et al., 2019) that can be used to gain novel insights into GA and potentially as a tool for drug development.

Early stages of geographic atrophy (GA) age-related macular degeneration is characterised by the demise of photoreceptors, which precedes the loss of underlying retinal pigment epithelial (RPE) cells. Sight-loss due to GA has no effective treatment; reflecting both the complexity of the disease and the lack of suitable animal models for testing potential therapies. We report the development and characterisation of a laser-induced mouse model with early GA-like pathology. Retinas were lasered at adjacent sites using a 810 nm laser (1.9 J/spot), resulting in the development of confluent, hypopigmented central lesions with well-defined borders. Optical Coherence Tomography over 2-months showed progressive obliteration of photoreceptors with hyper-reflective outer plexiform and RPE/Bruch’s membrane (BrM) layers within lesions, but an unaffected inner retina. Light/electron microscopy after 3-months revealed lesions without photoreceptors, leaving the outer plexiform layer apposed to the RPE. We observed outer segment debris, hypo/hyperpigmented RPE, abnormal apical-basal RPE surfaces and BrM thickening. Lesions had wedge-shaped margins, extended zones of damage, activated Müller cells, microglial recruitment and functional retinal deficits. mRNA studies showed complement and inflammasome activation, microglial/macrophage phagocytosis and oxidative stress providing mechanistic insights into GA. We propose this mouse model as an attractive tool for early GA studies and drug-discovery.

The transport and targeting of internalized molecules to distinct intracellular organelles/compartments can prove challenging to visualize clearly, which can contribute to some of the difficulties associated with these studies. By combining several approaches, we show how the trafficking and processing of photoreceptor outer segments in the phagosome and autophagy-lysosomal pathways of the retinal pigment epithelium (RPE) can easily be quantified and visualized as 3D-reconstructed images. This protocol takes advantage of new developments in microscopy and image-analysis software which has the potential to help better understand dynamic intracellular processes that underlie RPE dysfunction associated with irreversible blinding diseases such as age-related macular degeneration. The method described herein can also be used to study the trafficking and co-localization of different intracellular cargos in other cell types and tissues.

The transport and targeting of internalized molecules to distinct intracellular organelles/compartments can prove challenging to visualize clearly, which can contribute to some of the difficulties associated with these studies. By combining several approaches, we show how the trafficking and processing of photoreceptor outer segments in the phagosome and autophagy-lysosomal pathways of the retinal pigment epithelium (RPE) can easily be quantified and visualized as 3D-reconstructed images. This protocol takes advantage of new developments in microscopy and image-analysis software which has the potential to help better understand dynamic intracellular processes that underlie RPE dysfunction associated with irreversible blinding diseases such as age-related macular degeneration. The method described herein can also be used to study the trafficking and co-localization of different intracellular cargos in other cell types and tissues.

Age-related macular degeneration (AMD) is the leading cause of irreversible central vision loss, typically affecting individuals from mid-life onwards. Its multifactorial aetiology and the lack of any effective treatments has spurred the development of animal models as research and drug discovery tools. Several rodent models have been developed which recapitulate key features of AMD and provide insights into its underlying pathology. These have contributed to making significant progress in understanding the disease and the identification of novel therapeutic targets. However, a major caveat with existing models is that they do not demonstrate the full disease spectrum. In this review, we outline advances in rodent AMD models from the last decade. These models feature various hallmarks associated with AMD, including oxidative stress, hypoxia, immune dysregulation, genetic mutations and environmental risk factors. The review summarises the methods by which each model was created, its pathological characteristics as well as its relation to the disease in humans.

Impaired cargo trafficking and the aggregation of intracellular macromolecules are key features of neurodegeneration, and a hallmark of aged as well as diseased retinal pigment epithelial (RPE) cells in the eye. Here, photoreceptor outer segments (POS), which are internalized daily by RPE cells, were modified by UV-irradiation to create oxidatively modified POS (OxPOS). Oxidative modification was quantified by a protein carbonyl content assay. Human ARPE-19 cells were synchronously pulsed with POS or OxPOS to study whether oxidatively modified cargos can recapitulate features of RPE pathology associated with blinding diseases. Confocal immunofluorescence microscopy analysis showed that OxPOS was trafficked to LAMP1, LAMP2 lysosomes and to LC3b autophagy vacuoles. Whilst POS were eventually degraded, OxPOS cargos were sequestered in late compartments. Co-localization of OxPOS was also associated with swollen autolysosomes. Ultrastructural analysis revealed the presence of electron-dense OxPOS aggregates in RPE cells, which appeared to be largely resistant to degradation. Measurement of cellular autofluorescence, using parameters used to assess fundus autofluorescence (FAF) in age-related macular disease (AMD) patients, revealed that OxPOS contributed significantly to a key feature of aged and diseased RPE. This in vitro cell model therefore represents a versatile tool to study disease pathways linked with RPE damage and sight-loss.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a component of the extracellular environment, where it mediates diverse processes including matrix regulation/turnover, inflammation and angiogenesis. Rare TIMP-3 risk alleles and mutations are directly linked with retinopathies such as age-related macular degeneration (AMD) and Sorsby fundus dystrophy, and potentially, through indirect mechanisms, with Alzheimer’s disease. Insights into TIMP-3 activities may be gleaned from studying Sorsby-linked mutations. However, recent findings do not fully support the prevailing hypothesis that a gain of function through the dimerisation of mutated TIMP-3 is responsible for retinopathy. Findings from Alzheimer’s patients suggest a hitherto poorly studied relationship between TIMP-3 and the Alzheimer’s-linked amyloid-beta (A) proteins that warrant further scrutiny. This may also have implications for understanding AMD as aged/diseased retinae contain high levels of A. Findings from TIMP-3 knockout and mutant knock-in mice have not led to new treatments, particularly as the latter does not satisfactorily recapitulate the Sorsby phenotype. However, recent advances in stem cell and in vitro approaches offer novel insights into understanding TIMP-3 pathology in the retina-brain axis, which has so far not been collectively examined. We propose that TIMP-3 activities could extend beyond its hitherto supposed functions to cause age-related changes and disease in these organs.

The retinal pigment epithelium (RPE) plays a key role in the pathogenesis of several blinding retinopathies. Alterations to RPE structure and function are reported in Age-related Macular Degeneration, Stargardt and Best disease as well as pattern dystrophies. However, the precise role of RPE cells in disease aetiology remains incompletely understood. Many studies into RPE pathobiology have utilised animal models, which only recapitulate limited disease features. Some studies are also difficult to carry out in animals as the ocular space remains largely inaccessible to powerful microscopes. In contrast,in-vitromodels provide an attractive alternative to investigating pathogenic RPE changes associated with age and disease. In this article we describe the step-by-step approach required to establish an experimentally versatilein-vitroculture model of the outer retina incorporating the RPE monolayer and supportive Bruch’s membrane (BrM). We show that confluent monolayers of the spontaneously arisen human ARPE-19 cell-line cultured under optimal conditions reproduce key features of native RPE. These models can be used to study dynamic, intracellular and extracellular pathogenic changes using the latest developments in microscopy and imaging technology. We also discuss how RPE cells from human foetal and stem-cell derived sources can be incorporated alongside sophisticated BrM substitutes to replicate the aged/diseased outer retina in a dish. The work presented here will enable users to rapidly establish a realisticin-vitromodel of the outer retina that is amenable to a high degree of experimental manipulation which will also serve as an attractive alternative to using animals. Thisin-vitromodel therefore has the benefit of achieving the 3Rs objective of reducing and replacing the use of animals in research. As well as recapitulating salient structural and physiological features of native RPE, other advantages of this model include its simplicity, rapid set-up time and unlimited scope for detailed single-cell resolution and matrix studies.

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular dystrophy with an estimated prevalence of 1 in 220,000 and an onset of disease around the 4th to 6th decade of life. Similar to age-related macular degeneration (AMD), ophthalmoscopy reveals accumulation of protein/lipid deposits under the retinal pigment epithelium (RPE), referred to as drusen, in the eyes of patients with SFD.SFD is caused by variants in the gene for tissue inhibitor of metalloproteinases-3 (TIMP3), which has been found in drusen-like deposits of SFD patients. TIMP3 is constitutively expressed by RPE cells and, in healthy eyes, resides in Bruch's membrane. Most SFD-associated TIMP3 variants involve the gain or loss of a cysteine residue. This suggests the protein aberrantly forms intermolecular disulphide bonds, resulting in the formation of TIMP3 dimers. It has been demonstrated that SFD-associated TIMP3 variants are more resistant to turnover, which is thought to be a result of dimerisation and thought to explain the accumulation of TIMP3 in drusen-like deposits at the level of Bruch's membrane.An important function of TIMP3 within the outer retina is to regulate the thickness of Bruch's membrane. TIMP3 performs this function by inhibiting the activity of matrix metalloproteinases (MMPs), which have the function of catalysing breakdown of the extracellular matrix. TIMP3 has an additional function to inhibit vascular endothelial growth factor (VEGF) signalling and thereby to inhibit angiogenesis. However, it is unclear whether SFD-associated TIMP3 variant proteins retain these functions. In this review, we discuss the current understanding of the potential mechanisms underlying development of SFD and summarise all known SFD-associated TIMP3 variants.Cell culture models provide an invaluable way to study disease and identify potential treatments. These allow a greater understanding of RPE physiology and pathophysiology, including the ability to study the blood-retinal barrier as well as other RPE functions such as phagocytosis of photoreceptor outer segments. This review describes some examples of such recent in vitro studies and how they might provide new insights into degenerative diseases like SFD.Thus far, most studies on SFD have been performed using ARPE-19 cells or other, less suitable, cell-types. Now, induced pluripotent stem cell (iPSC) technologies allow the possibility to non-invasively collect somatic cells, such as dermal fibroblast cells and reprogram those to produce iPSCs. Subsequent differentiation of iPSCs can generate patient-derived RPE cells that carry the same disease-associated variant as RPE cells in the eyes of the patient. Use of these patient-derived RPE cells in novel cell culture systems should increase our understanding of how SFD and similar macular dystrophies develop.

Although visual problems are reported by patients with Alzheimer’s disease and dementia, studies into this particular aspect of neuropathology are scarce. The growing awareness of complex pathological processes in the ageing retina and brain however, enables us to consider this from a new perspective. Here we discuss the latest findings on the wide-ranging visual defects experienced by those suffering from Alzheimer’s disease and dementia. We propose that events leading to chronic degeneration of the retina and the brain in fact share many striking similarities. In particular, we discuss the role of the Alzheimer’s-related Amyloid beta (A?) group of peptides; that has been shown to accumulate in senescent retinas, correlated with increased risk of retinal degeneration. The high photo-oxidative retinal environment creates ideal conditions for A? aggregation, evidenced by high A?-loads reported in aged and donor eyes from patients with Age-related Macular degeneration. Consequently, longitudinal and non-invasive retinal assessments may provide invaluable information on incipient pathology and disease progression in the retina as well as the senescent brain. Such insights may not only lead to identifying new pathogenic mechanisms in the retina with implications for understanding Alzheimer’s disease, but reveal the underlying causes of visual abnormalities reported in patients with dementia.

Intrinsic ageing of human skin is a subtle and gradual process that demonstrates few clinical or histological features until old age (70 years). Initial work indicates that aged skin is "retinoid sensitive" but there is little data on the role of retinoic acid receptors (RARs) or retinoid X receptors (RXRs) in skin ageing. As nuclear retinoid receptors have been implicated in ageing in rodents, we studied the distribution of these receptors in intrinsically aged as compared to young, photoprotected human skin. We found that intrinsic ageing of skin in vivo is accompanied by significant increases of RAR alpha mRNA and protein whereas other isoforms show no alteration with age. In vitro transfection of COS-1 cells with the RAR alpha gene induces expression of matrix metalloproteinase-1 (MMP-1), an enzyme known to play an active role in remodelling of the dermis in intrinsically aged and photoaged skin. Furthermore, addition of all-trans retinoic acid (RA) to cultures of RAR alpha-transfected COS-1 cells diminishes RAR alpha and returns levels of MMP-1 to those approaching baseline. These results demonstrate that intrinsic ageing of human skin is accompanied by significant elevation in the content of RAR alpha and that over-expression of RAR alpha influences expression of MMP-1, an important mediator of skin ageing.

Age-related macular degeneration (AMD) causes irreversible loss of central vision for which there is no effective treatment. Incipient pathology is thought to occur in the retina for many years before AMD manifests from midlife onwards to affect a large proportion of the elderly. Although genetic as well as non-genetic/environmental risks are recognized, its complex aetiology makes it difficult to identify susceptibility, or indeed what type of AMD develops or how quickly it progresses in different individuals. Here we summarize the literature describing how the Alzheimer's-linked amyloid beta (Aβ) group of misfolding proteins accumulate in the retina. The discovery of this key driver of Alzheimer's disease in the senescent retina was unexpected and surprising, enabling an altogether different perspective of AMD. We argue that Aβ fundamentally differs from other substances which accumulate in the ageing retina, and discuss our latest findings from a mouse model in which physiological amounts of Aβ were subretinally-injected to recapitulate salient features of early AMD within a short period. Our discoveries as well as those of others suggest the pattern of Aβ accumulation and pathology in donor aged/AMD tissues are closely reproduced in mice, including late-stage AMD phenotypes, which makes them highly attractive to study dynamic aspects of Aβ-mediated retinopathy. Furthermore, we discuss our findings revealing how Aβ behaves at single-cell resolution, and consider the long-term implications for neuroretinal function. We propose Aβ as a key element in switching to a diseased retinal phenotype, which is now being used as a biomarker for late-stage AMD.