Human eyes with exfoliation syndrome (XFS) exhibit a distinctive pattern of iris transillumination defects that are recapitulated in Lyst mutant mice carrying the beige allele. Here I present the identification and characterization of the B6-Lystbg-J mouse model of XFS, modifiers of Lyst mediated ocular phenotypes, mechanisms of intraocular pressure (IOP) pathology related to circadian rhythms, and mechanisms of iris transillumination defects in the B6-Lystbg-J mice. Clinical and histological analysis shows that the B6-Lystbg-J mice have multiple similarities to human XFS including: iris transillumination defects, production of an exfoliative-like material, and pronounced pigment dispersion. Despite these insults, Lyst mutation does not cause increased IOP or optic nerve damage within the context of a C57BL/6J genetic background. However, defects in the circadian rhythm regulation of IOP were identified. Sequence analysis identifies that the beige mutation is predicted to delete a single isoleucine from the WD40 domain of the LYST protein. I identified CSNK2B as a binding partner of LYST and showed that LYSTbg-J completely disrupts the interaction. CSNK2B function in regulating E-cadherin and β-catenin binding is subsequently disrupted. These results lead to a working hypothesis that aspects of the XFS phenotype involve LYST and CSNK2B pathways, likely influencing cell-cell adherens junctions. Epistasis experiments were used to test for genetic modifiers of Lyst, which demonstrated that albino Lyst mutant mice exhibited complete rescue of Lyst-dependent iris phenotypes. In a genetic background-driven approach, a DBA/2J strain of congenic mice was created. The DBA/2J background, which harbors multiple mutations influencing melanosomal-proteins, enhanced Lyst dependent iris phenotypes. Thus, both experimental approaches implicated melanosomes, a potential source of oxidative stress, as mechanistically contributory. Supporting a contributory role of oxidative damage, Lyst mutation resulted in genetic context sensitive changes in iris lipid hydroperoxide levels, being lowest in albino and highest in DBA/2J mice. These results identified an association between oxidative damage to lipid membranes and severity of Lyst-mediated phenotypes, uncovering a new mechanism contributing to pathophysiology involving LYST. In conclusion these results demonstrate that mutation of the Lyst gene can produce ocular features of human XFS and suggests that LYST or LYST-interacting genes may contribute to XFS.

Lesions in the human Lyst gene are associated with the lysosomal disorder Chediak Higashi Syndrome. The absence of Lyst causes the formation of enlarged lysosome related compartments in all cells. This defect results in severe immunodeficiency, neurological dysfunction, and ultimately in death. Despite decades of research, the mechanism for how these enlarged compartments arise is not well established. Two opposing models have been proposed for Lyst function. The fission model describes Lyst as a positive regulator of fission from lysosomal compartments, while the fusion model identifies Lyst as a negative regulator of fusion between lysosomes. To date, a consensus on which model is correct has not been reached. This thesis details my investigation of Lyst function using Dictyostelium discoideum. To establish a definitive model for the function of the Dictyostelium Lyst ortholog, LvsB, we used assays that distinguish between defects in vesicle fusion versus fission. We compared the phenotype of cells defective in LvsB with that of two known fission defect mutants ([mu]3 and WASH null mutants). The temporal localization characteristics of the post-lysosomal marker vacuolin, as well as vesicular acidity and fusion dynamics of LvsB null cells are distinct from those of both fission defect mutants. These distinctions are predicted by the fusion defect model and implicate LvsB as a negative regulator of vesicle fusion. This work also presents evidence that LvsB antagonizes the function of two fusion regulatory proteins, Rab14 and dLIP5. The Dictyostelium Rab14 GTPase is known to stimulate lysosome fusion, and here we implicate dLIP5 as a promoter of Rab14 activity. Constitutive activation of Rab14 increases vesicle fusion in wild type cells but not in dLIP5 mutant cells. Thus, Rab14 activity is dependent on dLIP5. Additionally, the aberrant vesicle morphology and fusion phenotypes of LvsB mutant cells are suppressed by expression of dominant inactive Rab14 or disruption of dLIP5. This suppression suggests that LvsB antagonizes Rab14 activity to negatively regulate vesicle fusion. These studies validate the fusion model for LvsB function and provide new insights into the relationships that dictate vesicle fusion regulation. By extension, we propose that Lyst negatively regulates vesicle fusion by antagonizing the activity of a RabGTPase. / text

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