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Merve Sen, Oksana Kutsyr, Sylvia Bolz, Tsui-Fen Chou, Ray Deshaies, Blanca Arango-Gonzalez, Marius Ueffing; Pharmacological interference in the VCP/ERAD/proteasome axis rescues photoreceptor degeneration in RhP23H. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4881.
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One of the most prevalent mutations in rhodopsin (Rh) causing autosomal dominant Retinitis Pigmentosa (adRP) RhP23H, results in its misfolding and its retention in the Endoplasmic Reticulum (ER). Valosin-containing protein (VCP) is an ATP driven molecular checkpoint at the ER before these proteins are released from the ER towards plasma membrane. Misfolded proteins are selectively identified, retained at the ER and cleared by a process called ER-associated degradation (ERAD). ERAD overload due to RhP23H misfolding causes cell death. As several pathways regulating protein homeostasis are associated with ERAD, we tested their specific impact on photoreceptor (PR) degeneration through pharmacological inhibition.
Organotypic cultures from RhP23H retinas of transgenic rats at PN9 cultured for 6 days were treated with different concentrations of pathway specific inhibitors: NMS-873 interferes with quality control mechanisms by VCP inhibition; the proteasome inhibitor Bortezomib (BO) suppresses proteasomal degradation; the mannosidase inhibitor Kifunensine (KIF) blocks processing of glycoproteins, and Geldanamycin (GA) inhibits the protein-folding mechanism in the ER. PR cell survival was assessed by counting cell rows and measuring the percentage of TUNEL-positive cells. Rh distribution was checked using a specific antibody.
VCP inhibitor NMS-873 caused strong and dose-dependent protection at 5µM (TUNEL; treated: 2,4%±0,2, control: 6,76%±0,6, P<0,002). Proteasome inhibition by BO significantly reduced the number of dying cells at 10µM (TUNEL; treated: 3,13%±0,3, control: 5,42%±0,6, P<0,05). The number of PR cell rows in the ONL was increased by both, NMS-873 (5µM; treated: 9,6±0,3, control: 6,06±0,6, P<0,05), and BO (10µM; treated: 9,37±0,6, control: 7,6±0,1, P<0,002). Only VCP inhibitor corrected aberrant inner segment distribution of Rh shifting it to physiological outer segment (OS). In contrary, KIF and GA turned out to accelerate PR cell degeneration.
Modulation of the VCP/ERAD/proteasome axis by inhibiting VCP and proteasome rescues PR cells in RhP23H. Inhibition of VCP activity establishes proper transport of Rh to the OS. These results strongly suggest that the VCP/ERAD/proteasome axis acts as a critical checkpoint in PR homeostasis whereas other pathways in ERAD that control protein integrity or proper folding of proteins do not.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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