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Abstract Given the absence of approved treatments for pathogenic variants in Peripherin-2 (PRPH2), it is imperative to identify a universally effective therapeutic target for PRPH2 pathogenic variants. To test the hypothesis that formation of the elongated discs in presence of PRPH2 pathogenic variants is due to the presence of the full complement of rhodopsin in absence of the required amounts of functional PRPH2. Here we demonstrate the therapeutic potential of reducing rhodopsin levels in ameliorating disease phenotype in knockin models for p.Lys154del (c.458-460del) and p.Tyr141Cys (c.422 A > G) in PRPH2. Reducing rhodopsin levels improves physiological function, mitigates the severity of disc abnormalities, and decreases retinal gliosis. Additionally, intravitreal injections of a rhodopsin-specific antisense oligonucleotide successfully enhance the physiological function of photoreceptors and improves the ultrastructure of discs in mutant mice. Presented findings shows that reducing rhodopsin levels is an effective therapeutic strategy for the treatment of inherited retinal degeneration associated with PRPH2 pathogenic variants.

Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity. Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4 weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8 weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.

Abstract Usher syndrome (USH) is the leading cause of combined deafness and blindness, with USH2A being the most prevalent form. The mechanisms responsible for this debilitating sensory impairment remain unclear. This study focuses on characterizing the auditory phenotype in a mouse model expressing the c.2290delG mutation in usherin equivalent to human frameshift mutation c.2299delG. Previously we described how this model reproduces patient’s retinal phenotypes. Here, we present the cochlear phenotype, showing that the mutant usherin, is expressed during early postnatal stages. The c.2290delG mutation results in a truncated protein that is mislocalized within the cell body of the hair cells. The knock-in model also exhibits congenital hearing loss that remains consistent throughout the animal’s lifespan. Structurally, the stereocilia bundles, particularly in regions associated with functional hearing loss, are disorganized. Our findings shed light on the role of usherin in maintaining structural support, specifically in longer inner hair cell stereocilia, during development, which is crucial for proper bundle organization and hair cell function. Overall, we present a genetic mouse model with cochlear defects associated with the c.2290delG mutation, providing insights into the etiology of hearing loss and offering potential avenues for the development of effective therapeutic treatments for USH2A patients.

The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) superfamily plays a pivotal role in cellular trafficking by facilitating membrane fusion events. These SNARE proteins, including syntaxins, assemble into complexes that actively facilitate specific membrane fusion events. Syntaxins, as integral components of the SNARE complex, play a crucial role in initiating and regulating these fusion activities. While specific syntaxins have been extensively studied in various cellular processes, including neurotransmitter release, autophagy and endoplasmic reticulum (ER)-to-Golgi protein transport, their roles in the retina remain less explored. This review aims to enhance our understanding of syntaxins’ functions in the retina by shedding light on how syntaxins mediate membrane fusion events unique to the retina. Additionally, we seek to establish a connection between syntaxin mutations and retinal diseases. By exploring the intricate interplay of syntaxins in retinal function and health, we aim to contribute to the broader comprehension of cellular trafficking in the context of retinal physiology and pathology.

The c.2299delG mutation in usherin causes loss of hearing and vision. Here, the authors show in a mouse model of this disease that the expression of mutant usherin leads to retinitis pigmentosa and structural defects in the photoreceptor cilium associated with mislocalization of VLGR1 and WHRN.

Visual signal transduction takes place within a stack of flattened membranous ‘discs’ enclosed within the light-sensitive photoreceptor outer segment. The highly curved rims of these discs, formed in the process of disc enclosure, are fortified by large hetero-oligomeric complexes of two homologous tetraspanin proteins, PRPH2 (a.k.a. peripherin-2 or rds) and ROM1. While mutations in PRPH2 affect the formation of disc rims, the role of ROM1 remains poorly understood. In this study, we found that the knockout of ROM1 causes a compensatory increase in the disc content of PRPH2. Despite this increase, discs of ROM1 knockout mice displayed a delay in disc enclosure associated with a large diameter and lack of incisures in mature discs. Strikingly, further increasing the level of PRPH2 rescued these morphological defects. We next showed that disc rims are still formed in a knockin mouse in which the tetraspanin body of PRPH2 was replaced with that of ROM1. Together, these results demonstrate that, despite its contribution to the formation of disc rims, ROM1 can be replaced by an excess of PRPH2 for timely enclosure of newly forming discs and establishing normal outer segment structure.

Ariboflavinosis is a pathological condition occurring as a result of riboflavin deficiency. This condition is treatable if detected early enough, but it lacks timely diagnosis. Critical symptoms of ariboflavinosis include neurological and visual manifestations, yet the effects of flavin deficiency on the retina are not well investigated. Here, using a diet induced mouse model of riboflavin deficiency, we provide the first evidence of how retinal function and metabolism are closely intertwined with riboflavin homeostasis. We find that diet induced riboflavin deficiency causes severe decreases in retinal function accompanied by structural changes in the neural retina and retinal pigment epithelium (RPE). This is preceded by increased signs of cellular oxidative stress and metabolic disorder, in particular dysregulation in lipid metabolism, which is essential for both photoreceptors and the RPE. Though many of these deleterious phenotypes can be ameliorated by riboflavin supplementation, our data suggests that some patients may continue to suffer from multiple pathologies at later ages. These studies provide an essential cellular and mechanistic foundation linking defects in cellular flavin levels with the manifestation of functional deficiencies in the visual system and paves the way for a more in-depth understanding of the cellular consequences of ariboflavinosis.

Dysregulation of retinal metabolism is emerging as one of the major reasons for many inherited retinal diseases (IRDs), a leading cause of blindness worldwide. Thus, the identification of a common regulator that can preserve or revert the metabolic ecosystem to homeostasis is a key step in developing a treatment for different forms of IRDs. Riboflavin (RF) and its derivatives (flavins), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are essential cofactors for a wide range of cellular metabolic processes; hence, they are particularly critical in highly metabolically active tissues such as the retina. Patients with RF deficiency (ariboflavinosis) often display poor photosensitivity resulting in impaired low-light vision. We have identified a novel retina-specific RF binding protein called retbindin (Rtbdn), which plays a key role in retaining flavin levels in the neural retina. This role is mediated by its specific localization at the interface between the neural retina and retinal pigment epithelium (RPE), which is essential for metabolite and nutrient exchange. As a consequence of this vital function, Rtbdn’s role in flavin utilization and metabolism in retinal degeneration is discussed. The principal findings are that Rtbdn helps maintain high levels of retinal flavins, and its ablation leads to an early-onset retinal metabolic dysregulation, followed by progressive degeneration of rod and cone photoreceptors. Lack of Rtbdn reduces flavin levels, forcing the neural retina to repurpose glucose to reduce the production of free radicals during ATP production. This leads to metabolic breakdown followed by retinal degeneration. Assessment of the role of Rtbdn in several preclinical retinal disease models revealed upregulation of its levels by several folds prior to and during the degenerative process. Ablation of Rtbdn in these models accelerated the rate of retinal degeneration. In agreement with these in vivo studies, we have also demonstrated that Rtbdn protects immortalized cone photoreceptor cells (661W cells) from light damage in vitro. This indicates that Rtbdn plays a neuroprotective role during retinal degeneration. Herein, we discussed the specific function of Rtbdn and its neuroprotective role in retinal metabolic homeostasis and its role in maintaining retinal health.