The long-term goal of this project is to understand how cells cope with mutant Rhodopsin-1 (Rh-1) proteins that underlie age-related retinal degeneration in the Drosophila model for Autosomal Dominant Retinitis Pigmentosa (ADRP). The Rh-1 alleles in this model impair the encoded protein's folding property and impose stress to the endoplasmic reticulum (ER). Healthy young cells exhibit robust ER stress-response mechanisms that allow the afflicted photoreceptors to survive until old age. On the other hand, old cells succumb to stress by activating cell death pathways, leading to age-related retinal degeneration. A better understanding of these pathways may allow the development of effective therapeutic strategies against this disease. Through our unique Drosophila-based approach, we identified a number of cellular response mechanisms to mutant Rh-1 that were unexpected, yet likely to play important roles in ADRP. Here, we propose three Specific Aims to investigate those mechanisms.
In Aim 1, we plan to examine how cells degrade misfolded rhodopsins to reduce stress in the ER. We had previously established that a central ubiquitin ligase involved in this process, HRD1, can significantly delay the course of retinal degeneration in the Drosophila ADRP model. HRD1 requires the cooperation of other proteins that help recognize and degrade misfolded Rh-1. Through a genetic screen, we identified a poorly characterized carboxypeptidase as one of the most potent factors that help reduce misfolded Rh-1 levels. Here, we propose to validate the intriguing idea that HRD1-mediated degradation of misfolded rhodopsin requires this carboxypeptidase of unknown function.
In Aim 2, we propose to determine the role of translational inhibitors in the ER stress response. Translational inhibition s a common outcome of cellular stress, and specifically in response to misfolded protein overload in the ER, cells activate the translational inhibitor PERK to reduce the burden on the ER protein folding system. Unexpectedly, we discovered that another translational inhibitor, 4E-BP, is induced downstream of PERK. As 4E-BP is known for its effects in enhancing stress resistance and prolonging lifespan, we plan to test the functional significance of 4E-BP induction in the ADRP model. Moreover, we propose to determine how 4E-BP protects cells against ER stress.
In Aim 3, we propose to study how ER stress induces cell death. Many cell death regulators localize to the mitochondrial outer membrane for their function, and we hypothesize that a specific cell death pathway conveys stress signals from the ER to the mitochondria. Through a genetic screen, we identified two Cyclin Dependent Kinases (CDKs) that are unexpectedly involved in this cell death signaling. Here, we propose to test the idea these CDKs form a linear pathway that promotes pro-apoptotic events at the mitochondrial outer membrane. The genes of interest here will be further examined for their roles in age-related retinal degeneration in a Drosophila model of ADRP. A successful outcome of this proposal is expected to bring conceptual advances to the three poorly understood areas of ER stress response that are directly related to the pathology of ADRP.

Public Health Relevance

Retinitis Pigmentosa (RP) is a genetic disorder that leads to a progressive loss of vision. Autosomal Dominant form of this disease is the most common type, most frequently caused by mutations in the rhodopsin gene. Here we propose to use the Drosophila model for ADRP to study how healthy cells respond to such stress caused by such mutant proteins, and how one can use that knowledge to delay the course of retinal degeneration in that model.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (BVS)
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Neuhold, Lisa
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New York University
Anatomy/Cell Biology
Schools of Medicine
New York
United States
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Vasudevan, Deepika; Ryoo, Hyung Don (2016) Detection of Cell Death in Drosophila Tissues. Methods Mol Biol 1419:131-44
Mollereau, B; Rzechorzek, N M; Roussel, B D et al. (2016) Adaptive preconditioning in neurological diseases - therapeutic insights from proteostatic perturbations. Brain Res 1648:603-616
Kang, Kwonyoon; Ryoo, Hyung Don; Park, Jung-Eun et al. (2015) A Drosophila Reporter for the Translational Activation of ATF4 Marks Stressed Cells during Development. PLoS One 10:e0126795
Wang, Lifen; Ryoo, Hyung Don; Qi, Yanyan et al. (2015) PERK Limits Drosophila Lifespan by Promoting Intestinal Stem Cell Proliferation in Response to ER Stress. PLoS Genet 11:e1005220
Jang, Bo-Yun; Ryoo, Hyung Don; Son, Jaekyoung et al. (2015) Role of Drosophila EDEMs in the degradation of the alpha-1-antitrypsin Z variant. Int J Mol Med 35:870-6
Ryoo, Hyung Don (2015) Drosophila as a model for unfolded protein response research. BMB Rep 48:445-53
Vasudevan, Deepika; Ryoo, Hyung Don (2015) Regulation of Cell Death by IAPs and Their Antagonists. Curr Top Dev Biol 114:185-208
Wang, Lifen; Zeng, Xiankun; Ryoo, Hyung Don et al. (2014) Integration of UPRER and oxidative stress signaling in the control of intestinal stem cell proliferation. PLoS Genet 10:e1004568
Zhang, Tianyi; Liao, Yang; Hsu, Fu-Ning et al. (2014) Hyperactivated Wnt signaling induces synthetic lethal interaction with Rb inactivation by elevating TORC1 activities. PLoS Genet 10:e1004357
Coelho, Dina S; Cairrão, Fatima; Zeng, Xiaomei et al. (2013) Xbp1-independent Ire1 signaling is required for photoreceptor differentiation and rhabdomere morphogenesis in Drosophila. Cell Rep 5:791-801

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