Expression of red/green-cone opsin mutants K82E, P187S, M273K result in unique pathobiological perturbations to cone structure and function.

Long-and middle-wavelength cone photoreceptors, which are responsible for our visual acuity and color vision, comprise ~95% of our total cone population and are concentrated in the fovea of our retina. Previously, we characterized the disease mechanisms of the L/M-cone opsin missense mutations N94K, W177R, P307L, R330Q and G338E, all of which are associated with congenital blue cone monochromacy (BCM) or color-vision deficiency. Here, we used a similar viral vector-based gene delivery approach in M-opsin knockout mice to investigate the pathogenic consequences of the BCM or color-vision deficient associated L-cone opsin (OPN1LW) mutants K82E, P187S, and M273K. We investigated their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. K82E mutants were detected predominately in cone outer segments, and its expression partially restored expression and correct localization of cone PDE6α' and cone transducin γ. As a result, K82E also demonstrated the ability to mediate cone light responses. In contrast, expression of P187S was minimally detected by either western blot or by immunohistochemistry, probably due to efficient degradation of the mutant protein. M273K cone opsin appeared to be misfolded as it was primarily localized to the cone inner segment and endoplasmic reticulum. Additionally, M273K did not restore the expression of cone PDE6α' and cone transducin γ in dorsal cone OS, presumably by its inability to bind 11-cis retinal. Consistent with the observed expression pattern, P187S and M273K cone opsin mutants were unable to mediate light responses. Moreover, expression of K82E, P187S, and M273K mutants reduced cone viability. Due to the distinct expression patterns and phenotypic differences of these mutants observed in vivo, we suggest that the pathobiological mechanisms of these mutants are distinct.

Structural and functional rescue of cones carrying the most common cone opsin C203R missense mutation.

An arginine to cysteine substitution at amino acid position 203 (C203R) is the most common missense mutation in human cone opsin. Linked to color blindness and blue cone monochromacy (BCM), C203 is involved in a crucial disulfide bond required for proper folding. It has previously been postulated that expression of mutant C203R cone opsin exerts a toxic effect on cone photoreceptors, similar to some well-characterized missense mutations in rhodopsin that lead to protein misfolding. In this study, we generated and characterized a BCM mouse model carrying the equivalent C203R mutation (Opn1mwC198R Opn1sw-/-) to investigate the disease mechanism and develop a gene therapy approach for this disorder. Untreated Opn1mwC198R Opn1sw-/- cones phenocopied affected cones in human patients with the equivalent mutation, exhibiting shortened or absent cone outer segments and loss of function. We determined that gene augmentation targeting cones specifically yielded robust rescue of cone function and structure when Opn1mwC198R Opn1sw-/- mice were treated at early ages. Importantly, treated cones displayed elaborated outer segments and replenished expression of crucial cone phototransduction proteins. Interestingly, we were unable to detect OPN1MWC198R mutant opsin at any age. We believe this is the first proof-of-concept study exploring the efficacy of gene therapy in BCM associated with a C203R mutation.

A Modified Acyl-RAC Method of Isolating Retinal Palmitoyl Proteome for Subsequent Detection through LC-MS/MS.

Palmitoylation is a unique and reversible posttranslational lipid modification (PTM) that plays a critical role in many cellular events, including protein stability, activity, membrane association, and protein-protein interactions. The dynamic nature of palmitoylation dictates the efficient sorting of various retinal proteins to specific subcellular compartments. However, the underlying mechanism through which palmitoylation supports efficient protein trafficking in the retina remains unclear. Recent studies show that palmitoylation can also function as a signaling PTM, underlying epigenetic regulation and homeostasis in the retina. Efficient isolation of retinal palmitoyl proteome will pave the way to a better understanding of the role(s) for palmitoylation in visual function. The standard methods for detecting palmitoylated proteins employ 3H- or 14C-radiolabeled palmitic acid and have many limitations, including poor sensitivity. Relatively recent studies use thiopropyl Sepharose 6B resin, which offers efficient detection of palmitoylated proteome but is now discontinued from the market. Here, we describe a modified acyl resin-assisted capture (Acyl-RAC) method using agarose S3 high-capacity resin to purify palmitoylated proteins from the retina and other tissues, which is greatly compatible with downstream processing by LC-MS/MS. Unlike other palmitoylation assays, the present protocol is easy to perform and cost-effective. Graphical abstract.

Blue cone monochromacy and gene therapy.

Blue cone monochromacy (BCM) is a congenital vision disorder characterized by complete loss or severely reduced long- and middle-wavelength cone function, caused by mutations in the OPN1LW/OPN1MW gene cluster on the X-chromosome. BCM patients typically suffer from poor visual acuity, severely impaired color discrimination, myopia, and nystagmus. In this review, we cover the genetic causes of BCM, clinical features of BCM patients, genetic testing, and clinical outcome measurements for future BCM clinical trials. However, our emphasis is on detailing the animal models for BCM and gene therapy using adeno-associated vectors (AAV). We describe two mouse models resembling the two most common causes of BCM, current progress in proof-of-concept studies to treat BCM with deletion mutations, the challenges we face, and future directions.

R17C Mutation in Photoreceptor Disc-Specific Protein, PRCD, Results in Additional Lipidation Altering Protein Stability and Subcellular Localization.

Progressive rod-cone degeneration (PRCD) is a photoreceptor outer segment (OS) disc-specific protein essential for maintaining OS structures while contributing to rhodopsin packaging densities and distribution in disc membranes. Previously, we showed PRCD undergoing palmitoylation at the sole cysteine (Cys2), where a mutation linked with retinitis pigmentosa (RP) in humans and dogs demonstrates the importance of palmitoylation for protein stability and trafficking to the OS. We demonstrate a mutation, in the polybasic region (PBR) of PRCD (Arg17Cys) linked with RP where an additional lipidation is observed through acyl-RAC. Immunolocalization of transiently expressed R17C in hRPE1 cells depicts similar characteristics to wild-type PRCD; however, a double mutant lacking endogenous palmitoylation at Cys2Tyr with Arg17Cys is comparable to the C2Y protein as both aggregate, mislocalized to the subcellular compartments within the cytoplasm. Subretinal injection of PRCD mutant constructs followed by electroporation in murine retina exhibit mislocalization in the inner segment. Despite being additionally lipidated and demonstrating strong membrane association, the mutation in the PBR affects protein stability and localization to the OS. Acylation within the PBR alone neither compensates for protein stability nor trafficking, revealing defects in the PBR likely lead to dysregulation of PRCD protein associated with blinding diseases.

Gene Therapy in Opn1mw-/-/Opn1sw-/- Mice and Implications for Blue Cone Monochromacy Patients with Deletion Mutations.

Blue cone monochromacy (BCM) is a congenital vision disorder affecting both middle-wavelength (M) and long-wavelength (L) cone photoreceptors of the human retina. BCM results from abolished expression of green and red light-sensitive visual pigments expressed in M- and L-cones, respectively. Previously, we showed that gene augmentation therapy to deliver either human L- or M-opsin rescues dorsal M-opsin dominant cone photoreceptors structurally and functionally in treated M-opsin knockout (Opn1mw-/-) mice. Although Opn1mw-/- mice represent a disease model for BCM patients with deletion mutations, at the cellular level, dorsal cones of Opn1mw-/- mice still express low levels of S-opsin, which are different from L- and M-cones of BCM patients carrying a congenital opsin deletion. To determine whether BCM cones lacking complete opsin expression from birth would benefit from AAV-mediated gene therapy, we evaluated the outcome of gene therapy, and determined the therapeutic window and longevity of rescue in a mouse model lacking both M- and S-opsin (Opn1mw-/-/Opn1sw-/-). Our data show that cones of Opn1mw-/-/Opn1sw-/- mice are viable at younger ages but undergo rapid degeneration. AAV-mediated expression of human L-opsin promoted cone outer segment regeneration and rescued cone-mediated function when mice were injected subretinally at 2 months of age or younger. Cone-mediated function and visually guided behavior were maintained for at least 8 months post-treatment. However, when mice were treated at 5 and 7 months of age, the chance and effectiveness of rescue was significantly reduced, although cones were still present in the retina. Crossing Opn1mw-/-/Opn1sw-/- mice with proteasomal activity reporter mice (UbG76V-GFP) did not reveal GFP accumulation in Opn1mw-/-/Opn1sw-/- cones eliminating impaired degradation of ubiquitinated proteins as stress factor contributing to cone loss. Our results demonstrate that AAV-mediated gene augmentation therapy can rescue cone structure and function in a mouse model with a congenital opsin deletion, but also emphasize the importance that early intervention is crucial for successful therapy.

Nuclear NAD+-biosynthetic enzyme NMNAT1 facilitates development and early survival of retinal neurons.

Despite mounting evidence that the mammalian retina is exceptionally reliant on proper NAD+ homeostasis for health and function, the specific roles of subcellular NAD+ pools in retinal development, maintenance, and disease remain obscure. Here, we show that deletion of the nuclear-localized NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT1) in the developing murine retina causes early and severe degeneration of photoreceptors and select inner retinal neurons via multiple distinct cell death pathways. This severe phenotype is associated with disruptions to retinal central carbon metabolism, purine nucleotide synthesis, and amino acid pathways. Furthermore, transcriptomic and immunostaining approaches reveal dysregulation of a collection of photoreceptor and synapse-specific genes in NMNAT1 knockout retinas prior to detectable morphological or metabolic alterations. Collectively, our study reveals previously unrecognized complexity in NMNAT1-associated retinal degeneration and suggests a yet-undescribed role for NMNAT1 in gene regulation during photoreceptor terminal differentiation.

Loss of PRCD alters number and packaging density of rhodopsin in rod photoreceptor disc membranes.

Progressive rod-cone degeneration (PRCD) is a small protein localized to photoreceptor outer segment (OS) disc membranes. Several mutations in PRCD are linked to retinitis pigmentosa (RP) in canines and humans, and while recent studies have established that PRCD is required for high fidelity disc morphogenesis, its precise role in this process remains a mystery. To better understand the part which PRCD plays in disease progression as well as its contribution to photoreceptor OS disc morphogenesis, we generated a Prcd-KO animal model using CRISPR/Cas9. Loss of PRCD from the retina results in reduced visual function accompanied by slow rod photoreceptor degeneration. We observed a significant decrease in rhodopsin levels in Prcd-KO retina prior to photoreceptor degeneration. Furthermore, ultrastructural analysis demonstrates that rod photoreceptors lacking PRCD display disoriented and dysmorphic OS disc membranes. Strikingly, atomic force microscopy reveals that many disc membranes in Prcd-KO rod photoreceptor neurons are irregular, containing fewer rhodopsin molecules and decreased rhodopsin packing density compared to wild-type discs. This study strongly suggests an important role for PRCD in regulation of rhodopsin incorporation and packaging density into disc membranes, a process which, when dysregulated, likely gives rise to the visual defects observed in patients with PRCD-associated RP.