Date of Award

Fall 12-21-2018

Level of Access

Campus-Only Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

Advisor

Patsy M. Nishina

Second Committee Member

Gregory W. Carter

Third Committee Member

Gareth R. Howell

Additional Committee Members

Kenneth R. Johnson

Sandra Rieger

David S. Williams

Abstract

Retinal pigment epithelium (RPE) cells are specialized phagocytic cells that play critical roles in maintaining normal functions of the retina by engulfing and digesting the continuously shed photoreceptor outer segments (POS) and recycling the POS content. However, the regulatory mechanisms of phagocytosis, as a part of active vesicle transport in the RPE, are not clear. Although the common machineries of phagocytosis and other intracellular vesicle transport have been identified, new components of regulatory functions still await exploration. LYST (Lysosomal Trafficking regulator) is a ubiquitous protein implicated in lysosome and endosome fusion or fission. Disruption of LYST function results in Chediak-Higashi syndrome (CHS) in human populations, a rare autosomal recessive disorder characterized by recurrent fatal bacterial infection, neurological dysfunction and oculocutaneous albinism. The diagnostic feature of CHS is the enlargement of lysosomes and lysosome-related organelles (LROs) in all cell types examined, but the pathological mechanism underlying this clinical feature and the molecular function of LYST remain largely unclear. This work uses a novel mouse mutant with a disruption in Lyst, named rpea2 (retinal pigment epithelium atrophy 2), to study the previously undemonstrated roles of LYST in phagocytosis in RPE cells. We compared the number of rhodopsin-positive phagosomes within RPE histological sections and flat mount harvested from wild-type mice and rpea2 mutants, and showed a remarkable increase in the number of phagosomes in mutant RPE cells relative to wild-type controls. This indicates that the rpea2 mutation might impair or retard phagosome degradation and demonstrates that LYST plays an important role in the processing of phagosomes. Our work also suggests that the reduced phagosome degradation is at least in part due to decreased lysosomal hydrolytic activity and abnormal distribution of lysosomes. Investigating the regulation of phagocytosis in RPE cells, which are the most active phagocytic cells in the body, can also shed light on similar processes in other professional phagocytes, such as the phagocytes in the immune system. This is of paramount importance to the research of CHS, since the most life-threatening pathology of CHS patient is the recurrent bacterial infections. Thus, research on RPE phagocytosis can provide a unique opportunity to elucidate how LYST dysfunction may affect phagocytosis in vivo. Our data indicated, for the first time, that the Lystrpea2 mutation leads to increased expression level of ECM-remodeling proteases, cathepsin B, L S and MMP3, in RPE. In addition, we observed that mature active cathepsin B mainly localizes on the apical surface of RPE and around photoreceptor outer segments. The increase of cathepsin B is strongly correlated with the reduced adhesion observed between the RPE and the neural retina. In addition to cathepsin B, we also observed an elevated transcription level of cathepsin L and S in Lystrpea2 mutant RPE, and various studies have demonstrated that the overexpression of cathepsins is frequently accompanied by the secretion of pro-cathepsins. Cathepsin B, L and S are lysosomal cysteine proteases in normal cells and tissues, but it can be highly upregulated and becomes associated with the cell surface in malignant tumors and premalignant lesions. Our research also indicated an elevated transcriptional level of MMP3 in Lystrpea2 mutant RPE. MMP3 is a type of matrix metalloproteases (MMPs), which locates on cell membrane or in extracellular spaces and have been historically considered as essential actors of extracellular matrix (ECM) degradation. Cathepsin B, L, S and MMP3 are capable of remodeling the ECM, which is required for tumor cell invasion and metastasis. Thus, we propose that the increased level of cathepsin B, L, S and MMP3 may degrade the components of interphotoreceptor matrix (IPM) which lay between POS and RPE, and consequently causes the observed reduced association between RPE and neural retina, which predisposes rpea2 eyes to retinal detachment. In summary, understanding the function of LYST is important for developing effective therapies, not only for CHS but also diseases associated with alterations in LRO size and/or vesicular trafficking. Because cysteine cathepsins have been implicated in tumor cell invasion and metastasis, as well as in the pathogenesis of agerelated macular degeneration (AMD), study of Lystrpea2 RPE cells provide a unique opportunity to investigate the regulation of cysteine cathepsins and their contribution to tissue pathogenesis.

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