Abstract
AbstractPurposeOne 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). 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.MethodsOrganotypic cultures from RhP23H retinas of transgenic rats at PN9 cultured for 6 days and 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.ResultsVCP inhibitor caused strong and dose‐dependent protection at 5μM. Proteasome inhibition by BO significantly reduced the number of dying cells at 10μM. The number of PR cell rows in the ONL was increased by both. Only VCP inhibitor corrected aberrant inner segment distribution of Rh shifting it to physiological outer segment (OS). In contrary, KIF and GA accelerated PR cell degeneration.ConclusionsModulation 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.
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