Topographic differential analysis and function of age-related protein expression in the retinal pigment epithelium of Callithrix jacchus

The work presented here addresses the topographic differential analysis and function of the age-related protein expression in the retinal pigment epithelium (RPE) of Callithrix jacchus (C. jacchus). The modern medicine is responsible for an increased life span, with an increase of approximately three years in every generation with no evidence of an impending limit. The healthy, physiological cellular aging process is often accompanied by age-related dysfunctions which can lead to pathologies and affects many organs, including the eye. The conversion from the physiological aging process into a severe pathology is not fully understood. During the aging process of the eye, morphological as well as molecular changes in the retina as well as in the RPE can be observed. On a morphological basis, alterations occur in the intracellular granules in the RPE, including a decrease of unoccupied space in the cytoplasm of RPE cells, a lipofuscin redistribution by degranulation and enlargement and thickening of the RPE cell area. Additionally, the efficiency of some of the RPE functions, due to increased oxidative stress, are affected. Oxidative stress refers to a progressive cellular damage caused by reactive oxygen species (ROS) leading to protein misfolding and functional disbalance during cellular senescence. RPE cells digest photoreceptor outer segments, that are endocytosed and fused with lysosomes to be degraded. In aged RPE cells, lysosomal degradation is impaired resulting in accumulation of lipofuscin and protein damage, however the proteasomal and autophagy protein clearance systems are not working as effectively as in young RPE cells. The misfolded proteins are moved via exocytosis outside of the RPE cells. This material might be involved in drusen formation together with chronic inflammation and inflammatory cells. Drusen are a typical clinical sign of age-related macular degeneration. To understand the cellular mechanism of retinal aging, several analyses of the proteome of the retina in aging tissue were performed in the common marmoset C. jacchus and Sprague-Dawley rats to identify proteins for which the expression profiles are altered with maturation and aging. In the presented work, the whole transcriptome and the comparative analysis with the whole proteome of the RPE in aging C. jacchus was studied. Topological aspects regarding macular and peripheral parts of the RPE were considered to study age-related alterations. The analysis of the transcriptome revealed nearly 1500 genes whose expression significantly differed between macula and periphery in aged tissue, whereas only 114 genes were found to show such differences in neonatal tissue. Nearly 3000 genes are expressed significantly different in the neonatal macula versus the senile macula. These results are comparable with the proteomic data. Out of those data, the expression of several genes and proteins, including Nidogen 1, Thrombospondin 1 and Cathepsin B (CTSB), was analyzed and confirmed in further studies. Additionally, the presented work deals with the functional characterization of selected proteins, which were found in the transcriptomal analysis and previous studies regarding the aging retina in. One of these proteins is β-synuclein (SNCB). As an antagonist of -synuclein, its role in the neuronal system is partially investigated, but the role of SNCB in the aging RPE remains still unclear. Distinct age-related alterations of the topological and intracellular distribution of SNCB have been observed in the macula compared to periphery in the RPE of C. jacchus. In the macula, SNCB is mainly present in the cell membrane, whereas in the periphery it is mainly present in the cytoplasm and nucleus. For a functional characterization, ARPE-19 cells were incubated with recombinant SNCB. Decreased apoptosis rates, as well as an activation of the p53-pathway, promote senescence-related processes. Increased HMOX1 and NOX4 levels indicate an elevated oxidative stress and inflammatory response. The different distribution of SNCB in the primate RPE and alterations of cellular functions in SNCB-exposed cells promote its role in the aging RPE. The observation of an endorsed inflammatory response may indicate stress-related properties of SNCB. During the transcriptome analysis, CTSB was detected as a gene upregulated in aged tissue of C. jacchus. As it is involved in many functions correlating with the cellular aging process, it gained further research interest in this study. Comparable to SNCB, the function of CTSB is well investigated, but its role in the aging RPE is unknown. ARPE-19 cells exposed to recombinant CTSB show decreased cell viability and disrupted staining patterns of ZO-1 and Occludin, indicating a decreased integrity of intercellular tight junctions of RPE cells. Furthermore, a dose-depended alteration of the p53/MDM2 signaling cascade has been demonstrated. An elevated vascular endothelial growth factor expression indicates pro-angiogenic properties of recombinant CTSB-exposed RPE cells. ARPE-19 cells exposed to recombinant CTSB revealed alterations of relevant cellular conditions, like viability, intercellular tight junctions, and pro-angiogenic factors. Lastly, CTSB was introduced in a premature senescence model, to study the influence of CTSB on the RPE in-vitro. First, the premature senescence model was established in ARPE-19 cells using tert-butylhydroperoxide (tert-BHP). The activity of -galactosidase as well as oxidation of nucleic acids and lipids increased, indicating early senescence of ARPE-19 cells. The viability of the cells showed little change, while proliferation, the level of ROS and phagocytosis rate are significantly affected. While tert-BHP reduced proliferation, it increased intracellular ROS level and phagocytosis rate. The phagocytosis rate is also significantly increased by the additive exposure to tert-BHP and CTSB compared to the untreated control, whereas the apoptosis, proliferation and ROS production were not altered by CTSB. The study shows that in the model used early cellular senescence can be induced in-vitro. Furthermore, CTSB seems to selectively influence the functional behavior of senescent ARPE-19 cells. Summarized, this study gives a closer insight in the molecular aging process of the aging RPE in C. jacchus on gene as well as on protein level. By the number of genes whose expression differed significantly, one can conclude, that the macula is aging differently than the periphery, indicating that the functions of the single proteins involved in the aging process may differ between macula and periphery. Moreover, the functions of these proteins was mostly unclear. This study however gives an insight into the function of SNCB and CTSB in the (aging) RPE.


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