Photoreceptor Disc Enclosure Occurs in the Absence of Normal Peripherin-2/rds Oligomerization

Abstract

Mutations in the peripherin-2 gene (PRPH2, also known as rds) cause a heterogeneous range of autosomal dominant retinal diseases. PRPH2 encodes a photoreceptor-specific tetraspanin protein, PRPH2, that is a main structural component of the photoreceptor outer segment. PRPH2 distributes to the rims of outer segment disc membranes as they undergo the process of disc membrane enclosure. Within these rims, PRPH2 exists in homo-oligomeric form or as a hetero-oligomer with another tetraspanin protein, ROM1. While complete loss of PRPH2 prevents photoreceptor outer segment formation, mutations affecting the state of its oligomerization, including C150S, C213Y and Y141C, produce outer segment structural defects. In this study, we addressed whether any of these mutations also affect disc enclosure. We employed recently developed methodology for ultrastructural analysis of the retina, involving tissue processing with tannic acid, to assess the status of disc enclosure in knockin mouse models bearing either one or two alleles of the C150S, C213Y and Y141C PRPH2 mutations. While varying degrees of outer segment structural abnormalities were observed in each of these mouse models, they contained both newly forming "open" discs and mature "enclosed" discs. These data demonstrate that normal PRPH2 oligomerization is not essential for photoreceptor disc enclosure.

Keywords: disc; outer segment; peripherin; photoreceptor; retina.

Figure 1

Schematic representations of outer segment disc enclosure, peripherin-2 gene (PRPH2) oligomerization and PRPH2 molecular structure. (A) The base of a rod photoreceptor outer segment contains both “open,” nascent discs that are exposed to the extracellular space and “enclosed,” mature discs that are separated from the outer segment plasma membrane. In open discs, PRPH2 is located only at the discs rims adjacent to the connecting cilium. In enclosed discs, PRPH2 is located throughout the entire circumference of the disc. (B) The formation of PRPH2/ROM1 homo- and hetero-oligomers involves disulfide bonds between cysteines within the D2 loop of each molecule. (C) The molecular structure of PRPH2 is depicted with several features highlighted. Of note, the D2 loop contains seven cysteines: the C150 residue that is involved in intermolecular bonds for oligomerization and six that are involved in intramolecular bonds, including C213. This loop also contains the Y141 residue. Modified from Stuck et al. (2016) with permission by Elsevier.

Figure 2

Reduced expression of PRPH2 disrupts outer segment structure without preventing disc enclosure. Transmission electron microscopy (TEM) of photoreceptor outer segments from 12-day old WT (A) or rds/+ (B–D) mice stained with tannic acid. For each genotype, over 100 different outer segments from three mice were analyzed. For all images, arrowheads mark darkly stained open discs while arrows mark lightly stained enclosed discs. Scale bars are 1 μm.

Figure 3

Expression of C150S PRPH2 disrupts outer segment structure without preventing disc enclosure. (A–C) TEM of photoreceptor outer segments of 30-day old heterozygous C150S (C150S/+) mice stained with tannic acid. (D,E) TEM of photoreceptor outer segments of 16-day old C150S homozygous (C150S/C150S) mice stained with tannic acid. For each genotype, over 100 different outer segments from two mice were analyzed. For all images, arrowheads mark darkly stained open discs while arrows mark lightly stained enclosed discs. Scale bars are 1 μm.

Figure 4

Expression of C213Y PRPH2 disrupts outer segment structure without preventing disc enclosure. (A–C) TEM of photoreceptor outer segments of 30-day old heterozygous C213Y (C213Y/+) mice stained with tannic acid. Over 100 different outer segments from two mice were analyzed. For all images, arrowheads mark darkly stained open discs while arrows mark lightly stained enclosed discs. Scale bars are 1 μm.

Figure 5

Expression of Y141C PRPH2 disrupts outer segment structure without preventing disc enclosure. (A–C) TEM of photoreceptor outer segments of 90-day old heterozygous Y141C (Y141C/+) mice stained with tannic acid. (D–F) TEM of photoreceptor outer segments of 16-day old Y141C homozygous (Y141C/Y141C) mice stained with tannic acid. For each genotype, over 100 different outer segments from two mice were analyzed. For all images, arrowheads mark darkly stained open discs while arrows mark lightly stained enclosed discs. Scale bars are 1 μm.

Figure 6

Co-expression of Y141C and C150S PRPH2 disrupts outer segment structure without preventing disc enclosure. (A–D) TEM of photoreceptor outer segments of 30-day old compound heterozygous Y141C/C150S mice stained with tannic acid. For each genotype, over 100 different outer segments from two mice were analyzed. For all images, arrowheads mark darkly stained open discs while arrows mark lightly stained enclosed discs. Scale bars are 1 μm.