Doctor of Philosophy

dissertationFovea centralis is a depression located in the back of the primate retina. This region is crucial for sharp central vision. Age Related Macular Degeneration (AMD) is a debilitating disease that affects several million people in the world. In this disease, the retina and RPE surrounding the fovea undergoes degeneration, thereby compromising visual acuity. The fovea contains significant amounts of three carotenoids - lutein, zeaxanthin, and meso-zeaxanthin. Carotenoids are plant-derived pigment molecules. Vertebrates are unable to synthesize these compounds de novo, and have to obtain these through the diet. Carotenoids protect the foveal region from oxidative stress, light damage, and improves vision. Carotenoid supplementation is shown to alter the course of AMD. Hundreds of carotenoids are present in nature, and the regular human diet consumes about 50 of these. Among these, only 15 are absorbed by the gut and present in the serum. However, only lutein, zeaxanthin, and meso-zeaxanthin are present in the retina. Retinal concentrations of these carotenoids are 5000 times greater than observed levels in the serum, suggesting a very specific transport mechanism into the retina. There are gaps in our knowledge of the mechanisms involved in carotenoid transport into the eye. meso-Zeaxanthin is a retina-specific carotenoid that is rarely encountered in nature. No dietary sources of meso-zeaxanthin are identified. At the foveal pit, there are equal amounts of lutein, zeaxanthin, and meso-zeaxanthin. However, meso-zeaxanthin is absent in the peripheral retina. The mechanism by which meso-zeaxanthin is produced in the eye and the physiological significance of its presence is not understood

Impairment of Protein Trafficking upon Overexpression and Mutation of Optineurin

Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu(50) to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization.The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation

Biochemistry and Molecular Biology Developmentally Regulated Production of meso- Zeaxanthin in Chicken Retinal Pigment Epithelium/ Choroid and Retina

PURPOSE. meso-Zeaxanthin is a carotenoid that is rarely encountered in nature outside of the vertebrate eye. It is not a constituent of a normal human diet, yet this carotenoid comprises onethird of the primate macular pigment. In the current study, we undertook a systematic approach to biochemically characterize the production of meso-zeaxanthin in the vertebrate eye. METHODS. Fertilized White Leghorn chicken eggs were analyzed for the presence of carotenoids during development. Yolk, liver, brain, serum, retina, and RPE/choroid were isolated, and carotenoids were extracted. The samples were analyzed on C-30 or chiral HPLC columns to determine the carotenoid composition. RESULTS. Lutein and zeaxanthin were found in all studied nonocular tissues, but no mesozeaxanthin was ever detected. Among the ocular tissues, the presence of meso-zeaxanthin was consistently observed starting at embryonic day 17 (E17) in the RPE/choroid, several days before its consistent detection in the retina. If RPE/choroid of an embryo was devoid of mesozeaxanthin, the corresponding retina was always negative as well. CONCLUSIONS. This is the first report of developmentally regulated synthesis of mesozeaxanthin in a vertebrate system. Our observations suggest that the RPE/choroid is the primary site of meso-zeaxanthin synthesis. Identification of meso-zeaxanthin isomerase enzyme in the developing chicken embryo will facilitate our ability to determine the biochemical mechanisms responsible for production of this unique carotenoid in other higher vertebrates, such as humans

Autophagy in the eye:from physiology to pathophysology

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye

Cellular processing of myocilin.

Myocilin (MYOC) is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Pro370Leu (P370L) and Gln368stop (Q368X) have been identified in patients. In the present study, we investigated the processing of myocilin in human trabecular meshwork (TM) cells as well as in inducible, stable RGC5 cell lines.The turnover and photoactivation experiments revealed that the endogenous myocilin in human trabecular meshwork (TM) cells was a short-lived protein. It was found that the endogenous myocilin level in TM cells was increased by treatment of lysosomal and proteasomal inhibitors, but not by autophagic inhibitor. Multiple bands immunoreactive to anti-ubiquitin were seen in the myocilin pull down, indicating that myocilin was ubiquitinated. In inducible cell lines, the turnover rate of overexpressed wild-type and mutant P370L and Q368X myocilin-GFP fusion proteins was much prolonged. The proteasome function was compromised and autophagy was induced. A decreased PSMB5 level and an increased level of autophagic marker, LC3, were demonstrated.The current study provided evidence that in normal homeostatic situation, the turnover of endogenous myocilin involves ubiquitin-proteasome and lysosomal pathways. When myocilin was upregulated or mutated, the ubiquitin-proteasome function is compromised and autophagy is induced. Knowledge of the degradation pathways acting on myocilin can help in design of novel therapeutic strategies for myocilin-related glaucoma

Effects of inhibitors on levels of endogenous myocilin.

<p><b>A</b>. Western blotting. Human TM cells were treated for 16 h with vehicle DMSO or H₂O (not shown), or proteasomal (LCT and epoxomicin), lysosomal (NH₄Cl and chloroquin) or autophagic (3-MA) inhibitors. Proteins (25 µg) in cell lysates were immunoblotted with anti-myocilin or anti-GAPDH. Densitometry was performed. The myocilin/GAPDH relative to the DMSO control ratios are presented. <b>B</b>. Equal aliquots of media were immunoblotted with anti-myocilin. The level of myocilin in the media was normalized to that of the DMSO control. <b>C</b>. Immunofluorescence for myocilin. TM cells treated as above with the various inhibitors were fixed and immunostained for myocilin. All experiments were repeated at least 3 times, yielding similar results. Scale bar, 20 µm.</p

Effects of inhibitors on levels of expressed myocilin-GFP in inducible cells.

<p>Fluorescence images of myocilin_WT-GFP (Myoc_WT-GFP, <b>A</b>), myocilin_P370L-GFP (Myoc_P370L-GFP, <b>C</b>), myocilin_Q368X-GFP (Myoc_Q368X-GFP, <b>E</b>)-expressing-RGC5 cells treated with various inhibitors are shown. Scale bar, 20 µm. For Western blotting, RGC5 cells induced by Dox for 24 h to express myocilin_WT-GFP (<b>B</b>), myocilin_P370L-GFP (<b>D</b>), and myocilin_Q368X-GFP (<b>F</b>) were treated for 16 h with vehicle DMSO, or proteasomal (LCT and epoxomicin), lysosomal (NH₄Cl and chloroquin) or autophagic (3-MA) inhibitors. Proteins (25 µg) in cell lysates were immunoblotted with anti-myocilin or anti-GAPDH. Protein bands for the endogenous myocilin (55/57 kDa) and myocilin-GFP (∼100 kDa) were seen. Densitometry was performed. The myocilin-GFP/GAPDH relative to the DMSO control ratios are presented. All experiments were repeated at least 3 times.</p

Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease

Pušenje je štetna navika raširena među svim slojevima pučanstva. Smatra se rizičnim čimbenikom za zdravlje koji dovodi do prijevremenog umiranja. Duhanski dim sadrži više od četiri tisuće sastojaka koji djelujući podražajno, toksično, kancerogeno i psihoaktivno remete funkcije pojedinih organa. Ne postoji ni jedan organ ili organski sustav koji nije zahvaćen štetnim učincima pušenja. U ovom radu opisano je djelovanje duhanskog dima na krvožilni, dišni i probavni sustav. Također su spomenute i opasnosti pasivnog pušenja. Naglašena je važnost zdravstvenog odgoja te su razrađene metode zdravstvenog odgoja kojima se ovisnicima o pušenju nastoji promijeniti štetno zdravstveno ponašanje. Posebno je naglašena važnost medicinske sestre koja svojim znanjem i osobnim primjerom ima veliki utjecaj na smanjenje uporabe duhana kod pojedinca i zajednice. Opisan je pristup medicinske sestre kod određenih skupina kao što su trudnice, djeca i mladi te starije osobe.Smoking is a harmful habit widespread among all sections of the population. It is considered a risk factor for health leading to premature death. Tobacco smoke contains more than four thousand ingredients that have an irritant, toxic, carcinogenic and psychoactive effect on the functions of individual organs. There is no organ or organ system that is not affected by the harmful effects of smoking. This paper describes the effect of tobacco smoke on the circulatory, respiratory and digestive systems. The dangers of secondhand smoke were also mentioned. The importance of health education is emphasized and methods of health education that seek to change addictive behaviors are elaborated. Particular emphasis is placed on the importance of the nurse, who with her knowledge and personal example has a great influence on reducing the use of tobacco in individuals and in the community. The approach of the nurse to specific groups such as pregnant women, children and young people and the elderly is described

LC3 levels in inducible cells.

<p><b>A</b>. LC3 immunostaining (in red) in inducible, stable RGC5 cells. The cells were induced by Dox for 24 h to express myocilin_WT-GFP (MYOC), myocilin_P370L-GFP (P370L), or myocilin_Q368X-GFP (Q368X). The fusion protein-expressing, green fluorescent cells are shown in insets. There was no green fluorescence, as expected, in non-induced (control) cells. Note an increased LC3 staining intensity in myocilin-GFP-expressing green cells compared with non-induced cells. Scale bar, 10 µm. <b>B</b>. Western blotting for LC3 protein level. RGC5 cells were induced for 24 h to express myocilin_WT (Myoc_WT)-, myocilin_P370L (Myoc_P370L)-, or myocilin_Q368X (Myoc_Q368X)-GFP. Total cell lysate was subject to SDS-PAGE and immunoblotting using anti-LC3 or anti-GAPDH. Both LC3-I and LC3-II protein bands were detected. The LC3/GAPDH ratios in induced cells relative those of non-induced controls are presented. *, P<0.0046 compared to non-induced controls. All experiments were repeated at least 3 times, yielding similar results.</p