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

Lynn, Savannah A.; Ward, Gareth; Keeling, Eloise; Scott, Jenny A.; Cree, Angela J.; Johnston, D.; Page, Anton; Cuan-Urquizo, Enrique; Bhaskar, Atul; Grossel, Martin C.; Tumbarello, David A.; Newman, Tracey A.; Lotery, Andrew; Ratnayaka, J. Arjuna

doi:10.1016/j.tice.2017.06.003

Cited by 25 publications

(43 citation statements)

References 71 publications

(139 reference statements)

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“…In order to study how POS molecules were trafficked in the RPE we exploited a well‐characterized cell model which recapitulates key structural and physiological features of their native counterpart . Use of an in vitro system allowed a high degree of experimental manipulation as well as unimpeded access to high‐resolution microscopes and live cell imaging capabilities.…”

Section: Resultsmentioning

confidence: 99%

“…Such insights contribute to the current understanding of how cargos are trafficked and processed in the RPE. Although we and others have utilized well‐characterized approaches to ensure a tight pulse, the possibility of some premature POS internalization due to leakage cannot be fully excluded. However, it appears that a proportion of POS may be trafficked outside the conventional pathway via other mechanisms.…”

Section: Discussionmentioning

confidence: 99%

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Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE)

Keeling

Chatelet

Johnston

et al. 2019

Molecular Nutrition Food Res

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Scope Oxidative stress and dysregulated intracellular trafficking are associated with an unhealthy diet which underlies pathology. Here, these effects on photoreceptor outer segment (POS) trafficking in the retinal pigment epithelium (RPE), a major pathway of disease underlying irreversible sight‐loss, are studied. Methods and results POS trafficking is studied in ARPE‐19 cells using an algorithm‐based quantification of confocal‐immunofluorescence data supported by ultrastructural studies. It is shown that although POS are tightly regulated and trafficked via Rab5, Rab7 vesicles, LAMP1/2 lysosomes and LC3b‐autophagosomes, there is also a considerable degree of variation and flexibility in this process. Treatment with H2O2 and bafilomycin A1 reveals that oxidative stress and dysregulated autophagy target intracellular compartments and trafficking in strikingly different ways. These effects appear limited to POS‐containing vesicles, suggesting a cargo‐specific effect. Conclusion The findings offer insights into how RPE cells cope with stress, and how mechanisms influencing POS transport/degradation can have different outcomes in the senescent retina. These shed new light on cellular processes underlying retinopathies such as age‐related macular degeneration. The discoveries reveal how diet and nutrition can cause fundamental alterations at a cellular level, thus contributing to a better understanding of the diet‐disease axis.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE)

Keeling

Chatelet

Johnston

et al. 2019

Molecular Nutrition Food Res

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“…For example, accumulation of known drusen components such as vitronectin, amyloid beta, and clusterin has also been observed in the pores of the supporting membrane, whilst deposition of complement proteins has been demonstrated (Johnson et al, 2011;Lynn et al, 2017). Such advances in ex vivo cultures make them highly amenable to studying drusen formation as well as related aspects of RPE pathobiology.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Sorsby fundus dystrophy – A review of pathology and disease mechanisms

Christensen

Brown

Cree

et al. 2017

Experimental Eye Research

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143

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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.

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“…Of the several lysosomal enzymes mediating proteolytic breakdown of POS, Cathepsin D appears to be prominent in RPE cells 115 . This process of POS internalisation and cargo degradation can be studied using in-vitro cultures such as those utilised in our laboratory (Figure 3) 116,117 . ARPE-19 grown on transwell inserts for 2 months allowed cells to form confluent, hexagonal, pigmented monolayers expressing the cellspecific marker RPE65 and displaying apical and basolateral structural features of native RPE.…”

Section: Impairment Of Lysosomes and Other Components Of Cargo Handlimentioning

confidence: 99%

“…Desirable structural features and functional specialisation was similarly observed in our primary mouse RPE monolayers 117 cultured from postnatal day 10-12 C57BL/6 mice as reported by others 118,119 . Isolated POS were fluorescently labelled with Alexa Fluor FITC 488, which bound to MerTK and αvβ5 integrin receptors on the apical RPE surface 117 . Phagocytic ligands including MFG-E8 have been shown to be important for POS binding via the αvβ5 integrin receptor 44 .…”

Section: Impairment Of Lysosomes and Other Components Of Cargo Handlimentioning

confidence: 99%

Impaired Cargo Clearance in the Retinal Pigment Epithelium (RPE) Underlies Irreversible Blinding Diseases

Keeling¹,

Lotery²,

Tumbarello³

et al. 2018

Preprint

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Chronic degeneration of the Retinal Pigment Epithelium (RPE) is a precursor to pathological changes in the outer retina. The RPE monolayer, which lies beneath the neuroretina, daily internalises and digests large volumes of spent photoreceptor outer segments. Impaired cargo handling and processing in the endocytic/phagosome and autophagy pathways leads to the accumulation of lipofuscin and N-retinylidene-N-retinylethanolamine aggregates and chemically-modified compounds such as malondialdehyde and 4-hydroxynonenal within RPE. These contribute to increased proteolytic and oxidative stress, resulting in irreversible damage to post-mitotic RPE cells and development of blinding conditions such as Age-related Macular Degeneration, Stargardt disease and Choroideremia. Here, we review how impaired cargo handling in the RPE results in their dysfunction, discuss new findings from our laboratory and consider how newly discovered roles for lysosomes and the autophagy pathway could provide insights into retinopathies. Studies of these dynamic, molecular events have also been spurred on by recent advances in optics and imaging technology. Mechanisms underpinning lysosomal impairment in other degenerative conditions including storage disorders, a-synuclein pathologies and Alzheimer’s disease are also discussed. Collectively, these findings help transcend conventional understanding of these intracellular compartments as simple waste disposal bags to bring about a paradigm shift in the way lysosomes are perceived.

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

Abstract: Graphical abstract

Cited by 25 publications

References 71 publications

Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE)

Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE)

Sorsby fundus dystrophy – A review of pathology and disease mechanisms

Impaired Cargo Clearance in the Retinal Pigment Epithelium (RPE) Underlies Irreversible Blinding Diseases

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