J. Arjuna Ratnayaka

The complexities underlying age-related macular degeneration: could amyloid beta play an important role?

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 Alzheimers-linked amyloid beta (Aβ) group of misfolding proteins accumulate in the retina. The discovery of this key driver of Alzheimers 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.

Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE

Graphical abstract

The Alzheimer's-related amyloid beta peptide is internalised by R28 neuroretinal cells and disrupts the microtubule associated protein 2 (MAP-2)

Age-related Macular Degeneration (AMD) is a common, irreversible blinding condition that leads to the loss of central vision. AMD has a complex aetiology with both genetic as well as environmental risks factors, and share many similarities with Alzheimers disease. Recent findings have contributed significantly to unravelling its genetic architecture that is yet to be matched by molecular insights. Studies are made more challenging by observations that aged and AMD retinas accumulate the highly pathogenic Alzheimers-related Amyloid beta (Aβ) group of peptides, for which there appears to be no clear genetic basis. Analyses of human donor and animal eyes have identified retinal Aβ aggregates in retinal ganglion cells (RGC), the inner nuclear layer, photoreceptors as well as the retinal pigment epithelium. Aβ is also a major drusen constituent; found correlated with elevated drusen-load and age, with a propensity to aggregate in retinas of advanced AMD. Despite this evidence, how such a potent driver of neurodegeneration might impair the neuroretina remains incompletely understood, and studies into this important aspect of retinopathy remains limited. In order to address this we exploited R28 rat retinal cells which due to its heterogeneous nature, offers diverse neuroretinal cell-types in which to study the molecular pathology of Aβ. R28 cells are also unaffected by problems associated with the commonly used RGC-5 immortalised cell-line, thus providing a well-established model in which to study dynamic Aβ effects at single-cell resolution. Our findings show that R28 cells express key neuronal markers calbindin, protein kinase C and the microtubule associated protein-2 (MAP-2) by confocal immunofluorescence which has not been shown before, but also calretinin which has not been reported previously. For the first time, we reveal that retinal neurons rapidly internalised Aβ1-42, the most cytotoxic and aggregate-prone amongst the Aβ family. Furthermore, exposure to physiological amounts of Aβ1-42 for 24 h correlated with impairment to neuronal MAP-2, a cytoskeletal protein which regulates microtubule dynamics in axons and dendrites. Disruption to MAP-2 was transient, and had recovered by 48 h, although internalised Aβ persisted as discrete puncta for as long as 72 h. To assess whether Aβ could realistically localise to living retinas to mediate such effects, we subretinally injected nanomolar levels of oligomeric Aβ1-42 into wildtype mice. Confocal microscopy revealed the presence of focal Aβ deposits in RGC, the inner nuclear and the outer plexiform layers 8 days later, recapitulating naturally-occurring patterns of Aβ aggregation in aged retinas. Our novel findings describe how retinal neurons internalise Aβ to transiently impair MAP-2 in a hitherto unreported manner. MAP-2 dysfunction is reported in AMD retinas, and is thought to be involved in remodelling and plasticity of post-mitotic neurons. Our insights suggest a molecular pathway by which this could occur in the senescent eye leading to complex diseases such as AMD.

Identification of a functionally significant tri-allelic genotype in the Tyrosinase gene ( TYR ) causing hypomorphic oculocutaneous albinism (OCA1B)

Oculocutaneous albinism (OCA) and ocular albinism (OA) are inherited disorders of melanin biosynthesis, resulting in loss of pigment and severe visual deficits. OCA encompasses a range of subtypes with overlapping, often hypomorphic phenotypes. OCA1 is the most common cause of albinism in European populations and is inherited through autosomal recessive mutations in the Tyrosinase (TYR) gene. However, there is a high level of reported missing heritability, where only a single heterozygous mutation is found in TYR. This is also the case for other OCA subtypes including OCA2 caused by mutations in the OCA2 gene. Here we have interrogated the genetic cause of albinism in a well phenotyped, hypomorphic albinism population by sequencing a broad gene panel and performing segregation studies on phenotyped family members. Of eighteen probands we can confidently diagnose three with OA and OCA2, and one with a PAX6 mutation. Of six probands with only a single heterozygous mutation in TYR, all were found to have the two common variants S192Y and R402Q. Our results suggest that a combination of R402Q and S192Y with a deleterious mutation in a ‘tri-allelic genotype’ can account for missing heritability in some hypomorphic OCA1 albinism phenotypes.

Sorsby fundus dystrophy: a review of pathology and disease mechanisms

Abstract 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 Bruchs 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 Bruchs membrane. An important function of TIMP3 within the outer retina is to regulate the thickness of Bruchs 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. HighlightsSorsby fundus dystrophy (SFD) is a dominant macular disease leading to blindness.We estimate the prevalence of SFD at approximately 1 in 220,000.SFD is driven by multiple different variations in TIMP‐3, which are summarised.Mechanisms by which variants in TIMP3 might lead to disease are discussed.We propose that patient‐derived stem cells may provide a new model to study SFD.

A convenient protocol for establishing a human cell culture model of the outer retina.

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-vitro models 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 versatile in-vitro culture 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 realistic in-vitro model 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. This in-vitro model 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.

Alzheimer’s-related amyloid beta peptide aggregates in the ageing retina: implications for sight loss and dementia

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.