Rhodopsin mislocalization drives ciliary dysregulation in a novel autosomal dominant retinitis pigmentosa knock-in mouse model.

Rhodopsin mislocalization encompasses various blind conditions. Rhodopsin mislocalization is the primary factor leading to rod photoreceptor dysfunction and degeneration in autosomal dominant retinitis pigmentosa (adRP) caused by class I mutations. In this study, we report a new knock-in mouse model that harbors a class I Q344X mutation in the endogenous rhodopsin gene, which causes rod photoreceptor degeneration in an autosomal dominant pattern.

Restoring retinal polyunsaturated fatty acid balance and retina function by targeting ceramide in AdipoR1-deficient mice.

Mutations in the adiponectin receptor 1 gene (AdipoR1) lead to retinitis pigmentosa and are associated with age-related macular degeneration. This study explores the effects of AdipoR1 gene deficiency in mice, revealing a striking decline in ω3 polyunsaturated fatty acids (PUFA), an increase in ω6 fatty acids, and elevated ceramides in the retina. The AdipoR1 deficiency impairs peroxisome proliferator-activated receptor α signaling, which is crucial for FA metabolism, particularly affecting proteins associated with FA transport and oxidation in the retina and retinal pigmented epithelium.

Drug Discovery Strategies for Inherited Retinal Degenerations.

Inherited retinal degeneration is a group of blinding disorders afflicting more than 1 in 4000 worldwide. These disorders frequently cause the death of photoreceptor cells or retinal ganglion cells. In a subset of these disorders, photoreceptor cell death is a secondary consequence of retinal pigment epithelial cell dysfunction or degeneration.

Aster proteins mediate carotenoid transport in mammalian cells.

Some mammalian tissues uniquely concentrate carotenoids, but the underlying biochemical mechanism for this accumulation has not been fully elucidated. For instance, the central retina of the primate eyes displays high levels of the carotenoids, lutein, and zeaxanthin, whereas the pigments are largely absent in rodent retinas. We previously identified the scavenger receptor class B type 1 and the enzyme β-carotene-oxygenase-2 (BCO2) as key components that determine carotenoid concentration in tissues.

A smartphone based method for mouse fundus imaging.

Noninvasive in vivo imaging of the mouse retina is essential for eye research. However, imaging the mouse fundus is challenging due to its small size and requires specialized equipment, maintenance, and training. These issues hinder the routine evaluation of the mouse retina.

RPE Cells Engulf Microvesicles Secreted by Degenerating Rod Photoreceptors.

Rhodopsin is mislocalized to the inner segment plasma membrane (IS PM) in various blinding disorders including autosomal-dominant retinitis pigmentosa caused by class I rhodopsin mutations. In these disorders, rhodopsin-laden microvesicles are secreted into the extracellular milieu by afflicted photoreceptor cells. Using a model expressing class I mutant rhodopsin or Na/K-ATPase (NKA) fused to Dendra2, we fluorescently labeled the microvesicles and found retinal pigment epithelial (RPE) cells are capable of engulfing microvesicles containing rhodopsin.

Protein Sorting in Healthy and Diseased Photoreceptors.

Rods and cones are retinal photoreceptor neurons required for our visual sensation. Because of their highly polarized structures and well-characterized processes of G protein-coupled receptor-mediated phototransduction signaling, these photoreceptors have been excellent models for studying the compartmentalization and sorting of proteins. Rods and cones have a modified ciliary compartment called the outer segment (OS) as well as non-OS compartments.

Disrupted Plasma Membrane Protein Homeostasis in a Model of Retinitis Pigmentosa.

Rhodopsin mislocalization is frequently observed in retinitis pigmentosa (RP) patients. For example, class I mutant rhodopsin is deficient in the VxPx trafficking signal, mislocalizes to the plasma membrane (PM) of rod photoreceptor inner segments (ISs), and causes autosomal dominant RP. Mislocalized rhodopsin causes photoreceptor degeneration in a manner independent of light-activation.

Müller glia phagocytose dead photoreceptor cells in a mouse model of retinal degenerative disease.

Retinitis pigmentosa is a devastating, blinding disorder that affects 1 in 4000 people worldwide. During the progression of the disorder, phagocytic clearance of dead photoreceptor cell bodies has a protective role by preventing additional retinal damage from accumulation of cellular debris. However, the cells responsible for the clearance remain unidentified.

A small molecule mitigates hearing loss in a mouse model of Usher syndrome III.

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844).

Impairment of Vision in a Mouse Model of Usher Syndrome Type III.

Purpose: The purpose of this study was to obtain an Usher syndrome type III mouse model with retinal phenotype.

Methods: Speed congenic method was used to obtain Clrn1 exon 1 knockout (Clrn1-/-) and Clrn1N48K knockin (Clrn1N48K/N48K) mice under A/J background. To study the retinal functions of these mice, we measured scotopic and photopic ERG responses.

Retinol dehydrogenase 8 and ATP-binding cassette transporter 4 modulate dark adaptation of M-cones in mammalian retina.

Key Points: This study explores the molecular mechanisms that regulate the recycling of chromophore required for pigment regeneration in mammalian cones. We report that two chromophore binding proteins, retinol dehydrogenase 8 (RDH8) and photoreceptor-specific ATP-binding cassette transporter (ABCA4) accelerate the dark adaptation of cones, first, directly, by facilitating the processing of chromophore in cones, and second, indirectly, by accelerating the turnover of chromophore in rods, which is then recycled and delivered to both rods and cones. Preventing competition with the rods by knocking out rhodopsin accelerated cone dark adaptation, demonstrating the interplay between rod and cone pigment regeneration driven by the retinal pigment epithelium (RPE).

Applications of phototransformable fluorescent proteins for tracking the dynamics of cellular components.

In the past few decades, fluorescent proteins have revolutionized the field of cell biology. Phototransformable fluorescent proteins are capable of changing their excitation and emission spectra after being exposed to specific wavelength(s) of light. The majority of phototransformable fluorescent proteins have originated from marine organisms.

Rhodopsin Trafficking and Mistrafficking: Signals, Molecular Components, and Mechanisms.

Rhodopsin is a seven-transmembrane G protein-coupled receptor (GPCR) and is the main component of the photoreceptor outer segment (OS), a ciliary compartment essential for vision. Because the OSs are incapable of protein synthesis, rhodopsin must first be synthesized in the inner segments (ISs) and subsequently trafficked across the connecting cilia to the OSs where it participates in the phototransduction cascade. Rapid turnover of the OS necessitates a high rate of synthesis and efficient trafficking of rhodopsin to the cilia.

Monitoring of rhodopsin trafficking and mistrafficking in live photoreceptors.

Outer segment (OS) directed trafficking is required for accomplishing the extremely high concentration of rhodopsin and explicitly high photon sensitivity of rod photoreceptor cells. Aberrant targeting of rhodopsin often leads to blinding disorders, due to various mechanisms causing rhodopsin mislocalization. Until recently, it has been challenging to monitor the dynamics of rhodopsin biogenesis and trafficking.

Organization of cGMP sensing structures on the rod photoreceptor outer segment plasma membrane.

A diffusion barrier segregates the plasma membrane of the rod photoreceptor outer segment into 2 domains; one which is optimized for the conductance of ions in the phototransduction cascade and another for disk membrane synthesis. We propose the former to be named "phototransductive plasma membrane domain," and the latter to be named "disk morphogenic plasma membrane domain." Within the phototransductive plasma membrane, cGMP-gated channels are concentrated in striated membrane features, which are proximally located to the sites of active cGMP production within the disk membranes.

Submembrane assembly and renewal of rod photoreceptor cGMP-gated channel: insight into the actin-dependent process of outer segment morphogenesis.

The photoreceptor outer segment (OS) is comprised of two compartments: plasma membrane (PM) and disk membranes. It is unknown how the PM renewal is coordinated with that of the disk membranes. Here we visualized the localization and trafficking process of rod cyclic nucleotide-gated channel α-subunit (CNGA1), a PM component essential for phototransduction.

Retrograde intraciliary trafficking of opsin during the maintenance of cone-shaped photoreceptor outer segments of Xenopus laevis.

Photoreceptor outer segments (OSs) are essential for our visual perception, and take either rod or cone forms. The cell biological basis for the formation of rods is well established; however, the mechanism of cone formation is ill characterized. While Xenopus rods are called rods, they exhibit cone-shaped OSs during the early process of development.

An unconventional secretory pathway mediates the cilia targeting of peripherin/rds.

It is unclear how unconventional secretion interplays with conventional secretion for the normal maintenance and renewal of membrane structures. The photoreceptor sensory cilium is recognized for fast membrane renewal, for which rhodopsin and peripherin/rds (P/rds) play critical roles. Here, we provide evidence that P/rds is targeted to the cilia by an unconventional secretion pathway.

Signals governing the trafficking and mistrafficking of a ciliary GPCR, rhodopsin.

Rhodopsin is a cilia-specific GPCR essential for vision. Rhodopsin mislocalization is associated with blinding diseases called retinal ciliopathies. The mechanism by which rhodopsin mislocalizes in rod photoreceptor neurons is not well understood.

The mechanosensory structure of the hair cell requires clarin-1, a protein encoded by Usher syndrome III causative gene.

Mutation in the clarin-1 gene (Clrn1) results in loss of hearing and vision in humans (Usher syndrome III), but the role of clarin-1 in the sensory hair cells is unknown. Clarin-1 is predicted to be a four transmembrane domain protein similar to members of the tetraspanin family. Mice carrying null mutation in the clarin-1 gene (Clrn1(-/-)) show loss of hair cell function and a possible defect in ribbon synapse.

Noninvasive multiphoton fluorescence microscopy resolves retinol and retinal condensation products in mouse eyes.

Multiphoton excitation fluorescence microscopy (MPM) can image certain molecular processes in vivo. In the eye, fluorescent retinyl esters in subcellular structures called retinosomes mediate regeneration of the visual chromophore, 11-cis-retinal, by the visual cycle. But harmful fluorescent condensation products of retinoids also occur in the retina.

Visualization of retinoid storage and trafficking by two-photon microscopy.

Vertebrate vision is maintained by the retinoid (visual) cycle, a complex enzymatic pathway that operates in the retina to regenerate the visual chromophore, 11-cis-retinal, a prosthetic group of rhodopsin that undergoes activation by light. Many different mutations in genes encoding retinoid cycle proteins can cause a variety of human blinding diseases. Two-photon microscopy is an evolving, non-invasive, and repetitive imaging technology that can be used to monitor biomolecules within the vertebrate retina at a subcellular resolution.

Proteomic changes in the photoreceptor outer segment upon intense light exposure.

Acute light-induced photoreceptor degeneration has been studied in experimental animals as a model for photoreceptor cell loss in human retinal degenerative diseases. Light absorption by rhodopsin in rod photoreceptor outer segments (OS) induces oxidative stress and initiates apoptotic cell death. However, the molecular events that induce oxidative stress and initiate the apoptotic cascade remain poorly understood.

Clarin-1, encoded by the Usher Syndrome III causative gene, forms a membranous microdomain: possible role of clarin-1 in organizing the actin cytoskeleton.

Clarin-1 is the protein product encoded by the gene mutated in Usher syndrome III. Although the molecular function of clarin-1 is unknown, its primary structure predicts four transmembrane domains similar to a large family of membrane proteins that include tetraspanins. Here we investigated the role of clarin-1 by using heterologous expression and in vivo model systems.