טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentSafuri Shadi
SubjectPathogenesis of Macular Degeneration: The mechanism of
Lipofuscin Accumulation in a Model of Best
Disease, and the Distinct Transcriptome
of RPE Cells Facing Cone...
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ido Perlman
Full Thesis textFull thesis text - English Version


Abstract

Background and Rationale: Macular-degeneration is a group of diseases characterized with loss of central vision and decline visual acuity while visual field remains intact. Best-Disease is an inherited form caused by mutation in Bestrophin-1 (Best1). The expression of this protein is limited to the retinal pigment epithelium (RPE). Despite established biophysical functions, the mechanism/s leading to Best-Disease are yet unknown. Moreover, it is unclear why manifestation of the lesion is restricted to the macula, while the mutated Best1 is present in the entire RPE layer. Lipofuscin accumulation in the RPE suggests involvement of photoreceptor outer segments (POS) in the pathogenesis. Accordingly, the effects of Best1 mutations on phagocytosis of POS by ARPE-19 cells were tested. Additionally, in search for factors that can explain macular susceptibility in maculo-degenerative disorders, the transcriptome of RPE cells facing cone photoreceptors was compared to that of RPE facing rod photoreceptors.

Methods: ARPE-19 cell lines, stably expressing variants of Best1, were generated using site-directed-mutagenesis and retroviral transduction. Monolayers of these lines were challenged with FITC-labeled POS. Binding and engulfment were assessed. In search for factors that may explain macular susceptibility to RPE abnormalities, RNA was extracted from RPE cells of wild-type mice (rich in rods) and Nrl knock-out mice (rich in cones). Genome-wide comparison was made and expression of genes with interest was confirmed using Nanostring-nCounter technology.

Results: Following a saturating pulse of POS, ARPE-19 lines expressing Best-Disease associated mutants (R218S, E300D) displayed enhanced phagocytosis, while V86M mutation, associated with Autosomal Dominant Vitreoretinochoroidopathy (ADVIRC), showed a reduction in phagocytosis compared to wild-type Best1. Null-line, serving as retroviral transduction control, also displayed enhanced phagocytosis. The differences between wild-type and Best Disease lines, but not ADVIRC, were sensitive to DIDS and ATPγS. DIDS, a chloride channel blocker, diminished these differences. ATPγS, a stimulator of chloride current, enhanced them.  Buffering intracellular calcium with BAPTA-AM, reduced the difference in phagocytosis between ADVIRC and other lines. Comparing the transcriptome of RPE from wild-type and Nrl knock-out mice revealed up-regulation of several genes of interest in RPE from Nrl knock-out mice facing cones including genes coding for proteins participating in the phagocytosis process, complement, cellular adhesions, oxidation protection, and retinol metabolism.  Following the observation of higher levels of nicotinic receptor subunits transcripts, CHRNA3 and CHRNB4, nicotine was given in drinking water for 1 week. Significant increase in VEGF levels was observed in nicotine-treated Nrl knock-out mice but not in wild-types. 

Conclusions: Best1 mutations modulate phagocytosis in a phenotype-dependent manner. Wild-type Best1 physiological role in RPE cells might be to fine tune phagocytosis to prevent transient accumulation of high levels of all-trans-retinal, a condition favoring the formation of A2E, the toxic major fluorophore of lipofuscin. Restriction of lesions to the macula might be attributed to its cone-rich nature. Distinct gene expression profile of RPE cells facing cones can explain, at least partially, macular susceptibility to age related lesions, including genes coding for proteins of the complement system and cholinergic-receptors mediating nicotine effect on neovascularization in the cone-rich macula.