Date of Award

January 2014

Document Type


Degree Name

Medical Doctor (MD)



First Advisor

Lawrence J. Rizzolo

Second Advisor

Ron A. Adelman

Subject Area(s)

Ophthalmology, Biochemistry


Utility of Diverse Culture Models to Study how Autophagy Relates to the Pathogenesis of AMD. Haben F. Kefella, Ron A. Adelman, Lawrence J.Rizzolo, Department of Ophthalmology, New Haven CT

Age-Related Macular Degeneration (AMD) is a leading cause of blindness in the elderly in US. It affects over 1.7 million American's older than 65. Impaired autophagy has been implicated with lipofuscin accumulation that can lead to drusen formation and AMD. Our hypothesis is that autophagy capacity declines with age contributing to the pathogenesis of age-related macular degeneration. We examined autophagy in diverse culture models of RPE: secondary cultures of human fetal RPE (hfRPE), an ARPE-19 cell line, an immortalized cell line, and adult RPE donors (aged 50 and 90 years). All cultures were grown to confluence and maintained for 4-6 weeks in low or no-serum media. Autophagosome formation was assessed by using immunoblotting and immunofluorescence to monitor the conversion of LC3-I to LC3-II. Autophagy was either inhibited using Spautin-1, an inhibitor of beclin-1 or enhanced using rapamycin, an mTOR inhibitor. RNA sequencing and qRT-PCR was used to compare the expression of autophagy related genes. ARPE-19 and hfRPE expressed mRNA for various members of the autophagy pathway but in different relative amounts. Autophagic flux was higher in ARPE-19 and adult RPE (50yrs) compared to hfRPE and adult RPE (90yrs). Spautin-1 inhibited autophagy with an LC-50 ~10uM in ARPE-19. Spautin-1 required 12 to 24 hours to exert an effect. A spautin-1 dose of 10uM only partially inhibited autophagy in hfRPE as well as adult RPE. These cultures present contrasting models of autophagy in RPE. Autophagy appears more robust in ARPE-19 and adult RPE (50 yrs). The resistance of hfRPE and adult RPE to spautin-1 suggests an autophagy pathway that does not involve beclin-1. Learning to modulate autophagy in the RPE culture models may lead to therapies that stimulate autophagy in aged RPE.