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An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy.

ABSTRACT: 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.

SUBMITTER: Keeling E

PROVIDER: S-EPMC7555953 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy.

Keeling Eloise E Culling Annabelle J AJ Johnston David A DA Chatelet David S DS Page Anton A Tumbarello David A DA Lotery Andrew J AJ Ratnayaka J Arjuna JA

International journal of molecular sciences 20200911 18

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 P ...[more]

PMID: 32932802

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Project description:Aggregation of neuronal protein ?-synuclein leads to the formation of amyloid fibrils, which are associated with the development of Parkinson's disease. The mechanism of ?-synuclein pathology is not fully understood and is a subject of active research in the field. To tackle this problem, the fusions of fluorescent proteins to ?-synuclein C-terminus are often used in cellular and animal studies. The effects induced by such ?-synuclein sequence extension on ?-synuclein aggregation propensity are, however, not systematically examined despite the evidence that the negatively charged C-terminus plays a critical role in the regulation of ?-synuclein aggregation. In this work, we investigated how the charge and length variations of the C-terminus affect the aggregation propensity of ?-synuclein. To address these questions, we prepared mutants of ?-synuclein carrying additional moieties of different charge and length at the protein C-terminus. We determined the rates of two different aggregation stages (primary nucleation and elongation) based on a thioflavin T kinetic assay. We observed that all mutants bearing neutrally charged moieties of different length fibrilized slower than wild-type ?-synuclein. The primary nucleation and elongation rates strongly decreased with increase of the C-terminal extension length. Meanwhile, charge variation of the C-terminus significantly changed the rate of ?-synuclein nucleation, but did not markedly affect the rate of fibril elongation. Our data demonstrate that both the charge and length of the C-terminus play an important role at the stage of initial fibril formation, but the stage of fibril elongation is affected mainly by the length of C-terminal extension. In addition, our results suggest that there are at least two steps of incorporation of ?-synuclein monomers into the amyloid fibril: namely, the initial monomer binding to the fibril end (charge-dependent, relatively fast), and the subsequent conformational change of the protein (charge-independent, relatively slow, and thus the rate-limiting step).

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An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy.

Publications

An In-Vitro Cell Model of Intracellular Protein Aggregation Provides Insights into RPE Stress Associated with Retinopathy.

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