Comparative study of PRPH2 D2 loop mutants reveals divergent disease mechanism in rods and cones.

Mutations in the photoreceptor-specific tetraspanin gene peripherin-2 (PRPH2) lead to widely varying forms of retinal degeneration ranging from retinitis pigmentosa to macular dystrophy. Both inter- and intra-familial phenotypic heterogeneity has led to much interest in uncovering the complex pathogenic mechanisms of PRPH2-associated disease. Majority of disease-causing mutations in PRPH2 reside in the second intradiscal loop, wherein seven cysteines control protein folding and oligomerization. Here, we utilize knockin models to evaluate the role of three D2 loop cysteine mutants (Y141C, C213Y and C150S), alone or in combination. We elucidated how these mutations affect PRPH2 properties, including oligomerization and subcellular localization, and contribute to disease processes. Results from our structural, functional and molecular studies revealed that, in contrast to our understanding from prior investigations, rods are highly affected by PRPH2 mutations interfering with oligomerization and not merely by the haploinsufficiency associated with these mutations. On the other hand, cones are less affected by the toxicity of the mutant protein and significantly reduced protein levels, suggesting that knockdown therapeutic strategies may sustain cone functionality for a longer period. This observation provides useful data to guide and simplify the current development of effective therapeutic approaches for PRPH2-associated diseases that combine knockdown with high levels of gene supplementation needed to generate prolonged rod improvement.

Absence of retbindin blocks glycolytic flux, disrupts metabolic homeostasis, and leads to photoreceptor degeneration.

We previously reported a model of progressive retinal degeneration resulting from the knockout of the retina-specific riboflavin binding protein, retbindin (Rtbdn-/- ). We also demonstrated a reduction in neural retinal flavins as a result of the elimination of RTBDN. Given the role of flavins in metabolism, herein we investigated the underlying mechanism of this retinal degeneration by performing metabolomic analyses on predegeneration at postnatal day (P) 45 and at the onset of functional degeneration in the P120 retinas. Metabolomics of hydrophilic metabolites revealed that individual glycolytic products accumulated in the P45 Rtbdn-/- neural retinas along with the elevation of pentose phosphate pathway, while TCA cycle intermediates remained unchanged. This was confirmed by using 13C-labeled flux measurements and immunoblotting, revealing that the key regulatory step of phosphoenolpyruvate to pyruvate was inhibited via down-regulation of the tetrameric pyruvate kinase M2 (PKM2). Separate metabolite assessments revealed that almost all intermediates of acylcarnitine fatty acid oxidation, ceramides, sphingomyelins, and multiple toxic metabolites were significantly elevated in the predegeneration Rtbdn-/- neural retina. Our data show that lack of RTBDN, and hence reduction in flavins, forced the neural retina into repurposing glucose for free-radical mitigation over ATP production. However, such sustained metabolic reprogramming resulted in an eventual metabolic collapse leading to neurodegeneration.

Prph2 disease mutations lead to structural and functional defects in the RPE.

Prph2 is a photoreceptor-specific tetraspanin with an essential role in the structure and function of photoreceptor outer segments. PRPH2 mutations cause a multitude of retinal diseases characterized by the degeneration of photoreceptors as well as defects in neighboring tissues such as the RPE. While extensive research has analyzed photoreceptors, less attention has been paid to these secondary defects. Here, we use different Prph2 disease models to evaluate the damage of the RPE arising from photoreceptor defects. In Prph2 disease models, the RPE exhibits structural abnormalities and cell loss. Furthermore, RPE functional defects are observed, including impaired clearance of phagocytosed outer segment material and increased microglia activation. The severity of RPE damage is different between models, suggesting that the different abnormal outer segment structures caused by Prph2 disease mutations lead to varying degrees of RPE stress and thus influence the clinical phenotype observed in patients.

The Role of Peripherin-2/ROM1 Complexes in Photoreceptor Outer Segment Disc Morphogenesis.

The light-sensitive outer segment organelle of photoreceptor cells contains a stack of hundreds of flat, disc-shaped membranes called discs. The rims of these discs contain a photoreceptor-specific tetraspanin protein peripherin-2 (also known as rds or PRPH2). Mutations in the PRPH2 gene lead to a wide variety of inherited retinal degenerations in humans. The vast majority of these mutations occur within a large, intradiscal loop of peripherin-2, known as the D2 loop. The D2 loop mediates well-established intermolecular interactions of peripherin-2 molecules among themselves and a homologous protein ROM1. These interactions lead to the formation of large, highly ordered oligomers. In this chapter, we discuss the supramolecular organization of peripherin-2/ROM1 complexes and their contribution to the process of outer segment disc morphogenesis and enclosure.

Syntaxin 3 is essential for photoreceptor outer segment protein trafficking and survival.

Trafficking of photoreceptor membrane proteins from their site of synthesis in the inner segment (IS) to the outer segment (OS) is critical for photoreceptor function and vision. Here we evaluate the role of syntaxin 3 (STX3), in trafficking of OS membrane proteins such as peripherin 2 (PRPH2) and rhodopsin. Photoreceptor-specific Stx3 knockouts [Stx3f/f(iCre75) and Stx3f/f(CRX-Cre) ] exhibited rapid, early-onset photoreceptor degeneration and functional decline characterized by structural defects in IS, OS, and synaptic terminals. Critically, in the absence of STX3, OS proteins such as PRPH2, the PRPH2 binding partner, rod outer segment membrane protein 1 (ROM1), and rhodopsin were mislocalized along the microtubules to the IS, cell body, and synaptic region. We find that the PRPH2 C-terminal domain interacts with STX3 as well as other photoreceptor SNAREs, and our findings indicate that STX3 is an essential part of the trafficking pathway for both disc (rhodopsin) and rim (PRPH2/ROM1) components of the OS.

Photoreceptor Disc Enclosure Is Tightly Controlled by Peripherin-2 Oligomerization.

Mutations in the PRPH2 gene encoding the photoreceptor-specific protein PRPH2 (also known as peripherin-2 or rds) cause a broad range of autosomal dominant retinal diseases. Most of these mutations affect the structure of the light-sensitive photoreceptor outer segment, which is composed of a stack of flattened "disc" membranes surrounded by the plasma membrane. The outer segment is renewed on a daily basis in a process whereby new discs are added at the outer segment base and old discs are shed at the outer segment tip. New discs are formed as serial membrane evaginations, which eventually enclose through a complex process of membrane remodeling (completely in rods and partially in cones). As disc enclosure proceeds, PRPH2 localizes to the rims of enclosed discs where it forms oligomers which fortify the highly curved membrane structure of these rims. In this study, we analyzed the outer segment phenotypes of mice of both sexes bearing a single copy of either the C150S or the Y141C PRPH2 mutation known to prevent or increase the degree of PRPH2 oligomerization, respectively. Strikingly, both mutations increased the number of newly forming, not-yet-enclosed discs, indicating that the precision of disc enclosure is regulated by PRPH2 oligomerization. Without tightly controlled enclosure, discs occasionally over-elongate and form large membranous "whorls" instead of disc stacks. These data show that the defects in outer segment structure arising from abnormal PRPH2 oligomerization are manifested at the stage of disc enclosure.SIGNIFICANCE STATEMENT The light-sensitive photoreceptor outer segment contains a stack of flattened "disc" membranes that are surrounded, or "enclosed," by the outer segment membrane. Disc enclosure is an adaptation increasing photoreceptor light sensitivity by facilitating the diffusion of the second messenger along the outer segment axes. However, the molecular mechanisms by which photoreceptor discs enclose within the outer segment membrane remain poorly understood. We now demonstrate that oligomers of the photoreceptor-specific protein peripherin-2, or PRPH2, play an active role in this process. We further propose that defects in disc enclosure because of abnormal PRPH2 oligomerization result in major structural abnormalities of the outer segment, ultimately leading to loss of visual function and cell degeneration in PRPH2 mutant models and human patients.

ROM1 contributes to phenotypic heterogeneity in PRPH2-associated retinal disease.

Peripherin 2 (PRPH2) is a retina-specific tetraspanin protein essential for the formation of rod and cone photoreceptor outer segments (OS). Patients with mutations in PRPH2 exhibit severe retinal degeneration characterized by vast inter- and intra-familial phenotypic heterogeneity. To help understand contributors to this within-mutation disease variability, we asked whether the PRPH2 binding partner rod OS membrane protein 1 (ROM1) could serve as a phenotypic modifier. We utilized knockin and transgenic mouse models to evaluate the structural, functional and biochemical effects of eliminating one allele of Rom1 (Rom1+/-) in three different Prph2 models which mimic human disease: C213Y Prph2 (Prph2C/+), K153Del Prph2 (Prph2K/+) and R172W (Prph2R172W). Reducing Rom1 in the absence of Prph2 mutations (Rom1+/-) had no effect on retinal structure or function. However, the effects of reducing Rom1 in the presence of Prph2 mutations were highly variable. Prph2K/+/Rom1+/- mice had improved rod and cone function compared with Prph2K/+ as well as amelioration of K153Del-associated defects in PRPH2/ROM1 oligomerization. In contrast, Prph2R172W/Rom1+/- animals had worsened rod and cone function and exacerbated retinal degeneration compared with Prph2R172W animals. Removing one allele of Rom1 had no effect in Prph2C/+. Combined, our findings support a role for non-pathogenic ROM1 null variants in contributing to phenotypic variability in mutant PRPH2-associated retinal degeneration. Since the effects of Rom1 reduction are variable, our data suggest that this contribution is specific to the type of Prph2 mutation.

Prph2 initiates outer segment morphogenesis but maturation requires Prph2/Rom1 oligomerization.

The retinal disease gene peripherin 2 (PRPH2) is essential for the formation of photoreceptor outer segments (OSs), where it functions in oligomers with and without its homologue ROM1. However, the precise role of these proteins in OS morphogenesis is not understood. By utilizing a knock-in mouse expressing a chimeric protein comprised of the body of Rom1 and the C-terminus of Prph2 (termed RRCT), we find that the Prph2 C-terminus is necessary and sufficient for the initiation of OSs, while OS maturation requires the body of Prph2 and associated large oligomers. Importantly, dominant-negative physiological and biochemical defects in RRCT heterozygous rods are rescued by removing Rom1, suggesting Rom1 is a regulator for OS formation. Our experiments evaluating Prph2 trafficking show that Rom1 is a key determinant of whether Prph2 complexes utilize conventional versus unconventional (Golgi bypass) secretory pathways to reach the OS. These findings significantly advance our understanding of the molecular underpinnings of OS morphogenesis and particularly the role of Rom1.

Novel molecular mechanisms for Prph2-associated pattern dystrophy.

Mutations in peripherin 2 (PRPH2) have been associated with retinitis pigmentosa (RP) and macular/pattern dystrophies, but the origin of this phenotypic variability is unclear. The majority of Prph2 mutations are located in the large intradiscal loop (D2), a region that contains seven cysteines involved in intra- and intermolecular disulfide bonding and protein folding. A mutation at cysteine 213, which is engaged in an intramolecular disulfide bond, leads to butterfly-shaped pattern dystrophy in humans, in sharp contrast to mutations in the adjacent cysteine at position 214 which result in RP. To help understand this unexpected phenotypic variability, we generated a knockin mouse line carrying the C213Y disease mutation. The mutant Prph2 protein lost the ability to oligomerize with rod outer segment membrane protein 1 (Rom1), but retained the ability to form homotetramers. C213Y heterozygotes had significantly decreased overall Prph2 levels as well as decreased rod and cone function. Critically, supplementation with extra wild-type Prph2 protein elicited improvements in Prph2 protein levels and rod outer segment structure, but not functional rescue in rods or cones. These findings suggest that not all interruptions of D2 loop intramolecular disulfide bonding lead to haploinsufficiency-related RP, but rather that more subtle changes can lead to mutant proteins stable enough to exert gain-of-function defects in rods and cones. This outcome highlights the difficulty in targeting Prph2-associated gain-of-function disease and suggests that elimination of the mutant protein will be a pre-requisite for any curative therapeutic strategy.

Oligomerization of Prph2 and Rom1 is essential for photoreceptor outer segment formation.

Mutations in peripherin 2 (PRPH2, also known as Rds), a tetraspanin protein found in photoreceptor outer segments (OSs), cause retinal degeneration ranging from rod-dominant retinitis pigmentosa (RP) to cone-dominant macular dystrophy (MD). Understanding why some Prph2 mutants affect rods while others affect cones remains a critical unanswered question. Prph2 is essential for OS structure and function and exhibits a very specific pattern of oligomerization with its homolog Rom1. Non-covalent Prph2/Rom1 homo- and hetero-tetramers assemble into higher-order covalently linked complexes held together by an intermolecular disulfide bond at Prph2-C150/Rom1-C153. Here we disrupt this crucial bond using a C150S-Prph2 knockin mouse line to study the role of Prph2 higher-order complex formation. We find that C150S-Prph2 traffics to the OS, interacts with Rom1 and forms non-covalent tetramers, but alone cannot support normal OS structure and function. However, C150S-Prph2 supports the initiation or elaboration of OS disc structures, and improves rod OS ultrastructure in the presence of wild-type (WT) Prph2 (i.e. Prph2C150S/+ versus Prph2+/-). Prph2C150S/+ animals exhibit haploinsufficiency in rods, but a dominant-negative phenotype in cones, suggesting cones have a different requirement for large Prph2 complexes than rods. Importantly, cone but not rod function can be improved by the addition of one Prph2Y141C allele, a mutation responsible for pattern dystrophy owing to the extra cysteine. Combined these findings show that covalently linked Prph2 complexes are essential for OS formation, but not for Prph2 targeting to the OS, and that cones are especially sensitive to having a broad distribution of Prph2 complex types (i.e. tetramers and large complexes).

Retbindin: A riboflavin Binding Protein, Is Critical for Photoreceptor Homeostasis and Survival in Models of Retinal Degeneration.

The large number of inherited retinal disease genes (IRD), including the photopigment rhodopsin and the photoreceptor outer segment (OS) structural component peripherin 2 (PRPH2), has prompted interest in identifying common cellular mechanisms involved in degeneration. Although metabolic dysregulation has been shown to play an important role in the progression of the disease etiology, identifying a common regulator that can preserve the metabolic ecosystem is needed for future development of neuroprotective treatments. Here, we investigated whether retbindin (RTBDN), a rod-specific protein with riboflavin binding capability, and a regulator of riboflavin-derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is protective to the retina in different IRD models; one carrying the P23H mutation in rhodopsin (which causes retinitis pigmentosa) and one carrying the Y141C mutation in Prph2 (which causes a blended cone-rod dystrophy). RTBDN levels are significantly upregulated in both the rhodopsin (Rho)P23H/+ and Prph2Y141C/+ retinas. Rod and cone structural and functional degeneration worsened in models lacking RTBDN. In addition, removing Rtbdn worsened other phenotypes, such as fundus flecking. Retinal flavin levels were reduced in RhoP23H/+/Rtbdn-/- and Prph2Y141C/+/Rtbdn-/- retinas. Overall, these findings suggest that RTBDN may play a protective role during retinal degenerations that occur at varying rates and due to varying disease mechanisms.

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.

Oxidative Stress, Diabetic Retinopathy, and Superoxide Dismutase 3.

Diabetic retinopathy (DR) is a multifaceted disease, combining the deleterious effects of hyperglycemia and the propensity for accumulation of reactive oxygen species. Studies indicate that auto-oxidation of glucose, reduced antioxidant activity, and metabolic aberrations contribute to the pathogenesis of DR. These abnormalities stem from a fundamental imbalance between ROSs and antioxidant scavengers. To correct this imbalance and downstream effects, we propose that superoxide dismutase 3 (SOD3) is a viable therapeutic target for DR.

The Role of the Prph2 C-Terminus in Outer Segment Morphogenesis.

Peripherin 2 (also known as RDS/Prph2) is localized to the rims of rod and cone outer segment (OS) discs. The C-terminus of Prph2 is a critical functional domain, but its exact role is still unknown. In this mini review, we describe work on the Prph2 C-terminus, highlighting its role as a regulator of protein trafficking, membrane curvature, ectosome secretion, and membrane fusion. Evidence supports a role for the Prph2 C-terminus in these processes and demonstrates that it is necessary for the initiation of OS morphogenesis.

Flavin Imbalance as an Important Player in Diabetic Retinopathy.

The retina and RPE together constitute the most metabolically active ecosystem in the body, harboring high levels of flavins. Although diabetic patients have been reported to suffer from riboflavin deficiency and use of flavins as nutritional interventions to combat diabetic insult on other tissues have been investigated, such attempts have never been tested for the retina to avoid diabetic retinopathy. Furthermore, the role of flavins in pathophysiology of the retina and RPE has mostly been overlooked. Herein, we review the impact of flavins on various clinical manifestations of diabetic retinopathy and discuss possible ways to address them.

Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors.

Long-chain PUFAs (LC-PUFAs; C20-C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C 28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.

Ablation of the riboflavin-binding protein retbindin reduces flavin levels and leads to progressive and dose-dependent degeneration of rods and cones.

The interface between the neural retina and the retinal pigment epithelium (RPE) is critical for several processes, including visual pigment regeneration and retinal attachment to the RPE. One of its most important functions is the exchange of metabolites between the photoreceptors and RPE because photoreceptor cells have very high energy demands, largely satisfied by oxidative metabolism. The riboflavin (RF) cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), are two key cofactors involved in oxidative metabolism. We have previously shown that retbindin is a photoreceptor-specific RF-binding protein exclusively expressed in the rods and present in the interphotoreceptor matrix at the interface between the RPE and photoreceptor outer segments. Here, we show that retbindin ablation in mice causes a retinal phenotype characterized by time- and dose-dependent declines in rod and cone photoreceptor functions as early as 120 days of age. Whereas minor retinal ultrastructural defects were observed at all ages examined, a significant decline occurred in photoreceptor nuclei at 240 days of age (∼36.8% rods and ∼19.9% cones). Interestingly, significant reductions in FAD and FMN levels were observed before the onset of degeneration (∼46.1% FAD and ∼45% FMN). These findings suggest that the reduced levels of these flavins result in the disruption of intracellular mechanisms, leading to photoreceptor cell death. Altogether, our results suggest that retbindin is a key player in the acquisition and retention of flavins in the neural retina, warranting future investigation into retbindin's role in photoreceptor cell death in models of retinal degenerative disorders.

The K153Del PRPH2 mutation differentially impacts photoreceptor structure and function.

Peripherin 2 (Prph2) is a photoreceptor tetraspanin, and deletion of codon 153 (K153Δ) leads to retinitis pigmentosa, pattern dystrophy, and fundus flavimaculatus in the same family. To study this variability, we generated a K153Δ-Prph2 knockin mouse. K153Δ-Prph2 cannot form the complexes required for outer segment formation, and in cones cannot interact with its binding partner rod outer segment membrane protein 1. K153Δ causes dominant defects in rod and cone function; however, rod but not cone ultrastructure is improved by the presence of K153Δ-Prph2. Likewise, supplementation of K153Δ heterozygotes with WT-Prph2 results in structural but not functional improvements. These results support the idea that mutations may differentially affect Prph2's role as a structural component, and its role as a functional protein key for organizing membrane domains for cellular signalling. These roles may be different in rods and cones, thus contributing to the phenotypic heterogeneity that characterizes diseases associated with Prph2 mutations.

Phenotypic characterization of P23H and S334ter rhodopsin transgenic rat models of inherited retinal degeneration.

We produced 8 lines of transgenic (Tg) rats expressing one of two different rhodopsin mutations in albino Sprague-Dawley (SD) rats. Three lines were generated with a proline to histidine substitution at codon 23 (P23H), the most common autosomal dominant form of retinitis pigmentosa in the United States. Five lines were generated with a termination codon at position 334 (S334ter), resulting in a C-terminal truncated opsin protein lacking the last 15 amino acid residues and containing all of the phosphorylation sites involved in rhodopsin deactivation, as well as the terminal QVAPA residues important for rhodopsin deactivation and trafficking. The rates of photoreceptor (PR) degeneration in these models vary in proportion to the ratio of mutant to wild-type rhodopsin. The models have been widely studied, but many aspects of their phenotypes have not been described. Here we present a comprehensive study of the 8 Tg lines, including the time course of PR degeneration from the onset to one year of age, retinal structure by light and electron microscopy (EM), hemispheric asymmetry and gradients of rod and cone degeneration, rhodopsin content, gene dosage effect, rapid activation and invasion of the outer retina by presumptive microglia, rod outer segment disc shedding and phagocytosis by the retinal pigmented epithelium (RPE), and retinal function by the electroretinogram (ERG). The biphasic nature of PR cell death was noted, as was the lack of an injury-induced protective response in the rat models. EM analysis revealed the accumulation of submicron vesicular structures in the interphotoreceptor space during the peak period of PR outer segment degeneration in the S334ter lines. This is likely due to the elimination of the trafficking consensus domain as seen before as with other rhodopsin mutants lacking the C-terminal QVAPA. The 8 rhodopsin Tg lines have been, and will continue to be, extremely useful models for the experimental study of inherited retinal degenerations.