Rom1 converts Y141C-Prph2-associated pattern dystrophy to retinitis pigmentosa.

Mutations in peripherin 2 (PRPH2), also known as retinal degeneration slow/RDS, lead to various retinal degenerations including retinitis pigmentosa (RP) and macular/pattern dystrophy (MD/PD). PRPH2-associated disease is often characterized by a phenotypic variability even within families carrying the same mutation, raising interest in potential modifiers. PRPH2 oligomerizes with its homologue rod outer segment (OS) membrane protein 1 (ROM1), and non-pathogenic PRPH2/ROM1 mutations, when present together, lead to digenic RP. We asked whether ROM1 could modify the phenotype of a PRPH2 mutation associated with a high degree of intrafamilial phenotypic heterogeneity: Y141C. In vitro, Y141C-Prph2 showed signs of retention in the endoplasmic reticulum (ER), however co-expression with Rom1 rescued this phenotype. In the heterozygous Y141C knockin mouse model (Prph2Y/+), Y141C-Prph2 and Rom1 formed abnormal complexes but were present at normal levels. Abnormal complexes were eliminated in the absence of Rom1 (Prph2Y/+/Rom1-/-) and total Prph2 levels were reduced to those found in the haploinsufficient Prph2+/- RP model. The biochemical changes had functional and structural consequences; while Prph2Y/+ animals exhibited a cone-rod electroretinogram defect, Prph2Y/+/Rom1-/- animals displayed a rod-dominant phenotype and OSs similar to those seen in the Prph2+/-. These data show that ablation of Rom1 results in the conversion of an MD/PD phenotype characterized by cone functional defects and the formation of abnormal Prph2/Rom1 complexes to an RP phenotype characterized by rod-dominant functional defects and reductions in total Prph2 protein. Thus one method by which ROM1 may act as a disease modifier is by contributing to the large variability in PRPH2-associated disease phenotypes.

Dynamic Expression of Sox2, Gata3, and Prox1 during Primary Auditory Neuron Development in the Mammalian Cochlea.

Primary auditory neurons (PANs) connect cochlear sensory hair cells in the mammalian inner ear to cochlear nucleus neurons in the brainstem. PANs develop from neuroblasts delaminated from the proneurosensory domain of the otocyst and keep maturing until the onset of hearing after birth. There are two types of PANs: type I, which innervate the inner hair cells (IHCs), and type II, which innervate the outer hair cells (OHCs). Glial cells surrounding these neurons originate from neural crest cells and migrate to the spiral ganglion. Several transcription factors are known to regulate the development and differentiation of PANs. Here we systematically examined the spatiotemporal expression of five transcription factors: Sox2, Sox10, Gata3, Mafb, and Prox1 from early delamination at embryonic day (E) 10.5 to adult. We found that Sox2 and Sox10 were initially expressed in the proneurosensory cells in the otocyst (E10.5). By E12.75 both Sox2 and Sox10 were downregulated in the developing PANs; however, Sox2 expression transiently increased in the neurons around birth. Furthermore, both Sox2 and Sox10 continued to be expressed in spiral ganglion glial cells. We also show that Gata3 and Prox1 were first expressed in all developing neurons, followed by a decrease in expression of Gata3 and Mafb in type I PANs and Prox1 in type II PANs as they matured. Moreover, we describe two subtypes of type II neurons based on Peripherin expression. These results suggest that Sox2, Gata3 and Prox1 play a role during neurogenesis as well as maturation of the PANs.

Role of RDS and Rhodopsin in Cngb1-Related Retinal Degeneration.

PURPOSE: Rod photoreceptor outer segment (OS) morphogenesis, structural integrity, and proper signal transduction rely on critical proteins found in the different OS membrane domains (e.g., plasma, disc, and disc rim membrane). Among these key elements are retinal degeneration slow (RDS, also known as peripherin-2), rhodopsin, and the beta subunit of the cyclic nucleotide gated channel (CNGB1a), which have been found to interact in a complex. The purpose of this study was to evaluate the potential interplay between these three proteins by examining retinal disease phenotypes in animal models expressing varying amounts of CNGB1a, rhodopsin, and RDS. METHODS: Outer segment trafficking, retinal function, and photoreceptor structure were evaluated using knockout mouse lines. RESULTS: Eliminating Cngb1 and reducing RDS leads to additive defects in RDS expression levels and rod electroretinogram (ERG) function, (e.g., Cngb1-/-/rds+/- versus rds+/- or Cngb1-/-) but not to additive defects in rod ultrastructure. These additive effects also manifested in cone function: Photopic ERG responses were significantly lower in the Cngb1-/-/rds+/- versus rds+/- or Cngb1-/-, suggesting that eliminating Cngb1 can accelerate the cone degeneration that usually presents later in the rds+/-. This was not the case with rhodopsin; reducing rhodopsin levels in concert with eliminating CNGB1a did not lead to phenotypes more severe than those observed in the Cngb1 knockout alone. CONCLUSIONS: These data support a role for RDS as the core component of a multiprotein plasma membrane-rim-disc complex that has both a structural role in photoreceptor OS formation and maintenance and a functional role in orienting proteins for optimal signal transduction.

In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors.

Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod-specific PRPH2 mutations is rather characterized by PRPH2 downregulation and impaired protein localization.

Rim formation is not a prerequisite for distribution of cone photoreceptor outer segment proteins.

Retinal degeneration slow (RDS/PRPH2) is critical for the formation of the disc/lamella rim in photoreceptor outer segments (OSs), but plays a different role in rods vs. cones. Without RDS, rods fail to form OSs, however, cones lacking RDS (in the rds(-/-)/Nrl(-/-)) exhibit balloon-like OSs devoid of lamellae. We show that distribution of most proteins in the lamella and PM domains is preserved even in the absence of RDS, rim, and lamella structures. However, the rim protein prominin-1 exhibits altered trafficking and OS localization, suggesting that proper targeting and distribution of rim proteins may require RDS. Our ultrastructural studies show that in cones, OS formation is initiated by the growth of opsin-containing membrane with RDS-mediated rim formation as a secondary step. This is directly opposite to rods and significantly advances our understanding of the role of the rim in cone OS morphogenesis. Furthermore, our results suggest that the unique folded lamella architecture of the cone OS may maximize density or proximity of phototransduction proteins, but is not required for OS function or for protein distribution and retention in different membrane domains.