Loss of PRCD alters number and packaging density of rhodopsin in rod photoreceptor disc membranes

Abstract

Progressive rod-cone degeneration (PRCD) is a small protein localized to photoreceptor outer segment (OS) disc membranes. Several mutations in PRCD are linked to retinitis pigmentosa (RP) in canines and humans, and while recent studies have established that PRCD is required for high fidelity disc morphogenesis, its precise role in this process remains a mystery. To better understand the part which PRCD plays in disease progression as well as its contribution to photoreceptor OS disc morphogenesis, we generated a Prcd-KO animal model using CRISPR/Cas9. Loss of PRCD from the retina results in reduced visual function accompanied by slow rod photoreceptor degeneration. We observed a significant decrease in rhodopsin levels in Prcd-KO retina prior to photoreceptor degeneration. Furthermore, ultrastructural analysis demonstrates that rod photoreceptors lacking PRCD display disoriented and dysmorphic OS disc membranes. Strikingly, atomic force microscopy reveals that many disc membranes in Prcd-KO rod photoreceptor neurons are irregular, containing fewer rhodopsin molecules and decreased rhodopsin packing density compared to wild-type discs. This study strongly suggests an important role for PRCD in regulation of rhodopsin incorporation and packaging density into disc membranes, a process which, when dysregulated, likely gives rise to the visual defects observed in patients with PRCD-associated RP.

Dual Palmitoylation of PRCD, a Photoreceptor-Specific Protein Linked to RP, Alters Protein Stability and Subcellular Localization

“…We have demonstrated in an earlier study that palmitoylation deficiency in PRCD leads to severe protein destabilization and mistrafficking to the subcellular compartments in the photoreceptor IS (7,11). In rhodopsin, palmitoylation deficiency leads to visual impairment and light-induced photoreceptor degeneration (11,15,16,35). Furthermore, in small GTPase Ras protein, there are multiple lipidations in the C-terminal region along with the polybasic region essential for efficient protein trafficking to subcellular compartments and strong membrane anchoring (36)(37)(38).…”

“…PRCD has been shown to be associated with RP in humans and dogs, causing late-onset degeneration of retinal photoreceptor cells (5). There are six mutations in PRCD linked to RP, with the most common mutation being "cysteine 2 tyrosine" (C2Y) found in over 30 dog breeds and in humans (5,(7)(8)(9)(10)(11). The Prcd gene is expressed exclusively in retinal photoreceptor cells and encodes for a small 54 amino acid (aa) protein in humans and canines, and 53 aa protein in mice (Fig.…”

“…This information could divulge the precise role of PRCD in OS maintenance (12). As PRCD has been shown to be localized to the cytosolic surface of the outer segment (OS) disc membrane, although it does not appear to have a role in phototransduction exclusive presence in the disc membrane and its function remains widely unknown (11)(12)(13). Since the structure of the PRCD protein is not known, understanding the exclusive presence in the disc membrane is imperative.…”

“…We determined to study the role of the PBR in PRCD protein (19,(27)(28)(29)(30). Previous studies, including ours, demonstrated the strong association of PRCD to the cellular membrane despite the lack of palmitoylation (7,(11)(12)(13). To better assess the role of the PBR in PRCD protein stability and expression, we used GFP protein as an internal loading control for PRCD, which is driven independently by an internal ribosome entry site (IRES) (Fig.…”

“…PRCD is more concentrated at the base of the photoreceptor (PR) outer segment (OS) playing a critical role in OS disc morphogenesis (5,11,12). Earlier, our studies showed that PRCD lacking retina leads to defects in rhodopsin packaging density and distribution in the disc membranes (11). However, the connection between PRCD and rhodopsin packaging density in the disc membrane is not clearly understood.…”