Jul 2026· Investigative Ophthalmology and Visual Science· Vol 67, pp. 23· 0 citations· 59 references
Medicine
TL;DR
A pathogenic model in which elevated endocytosis and mitochondrial dysfunction contribute to the accelerated photoreceptor degeneration in RHO P347L-associated adRP is proposed.
Abstract
Purpose RHO mutations are the primary cause of autosomal dominant retinitis pigmentosa (adRP), with Class 1 mutations typically exhibiting more severe phenotypes than Class 2. This study aims to clarify the mechanistic basis for this clinical disparity by systematically comparing protein degradation pathways, mitochondrial stress, and neuroinflammation. Methods Humanized mouse lines carrying Class 1 (P347L) or Class 2 (L125R) RHO mutations were generated via CRISPR/Cas9-mediated knock-in. Retinal function, ultrastructure, and transcriptomic profiles were characterized through electroretinography (ERG), transmission electron microscopy (TEM), and RNA-sequencing (RNA-seq). To further elucidate molecular mechanisms, protein trafficking and degradation pathways were analyzed in transfected HEK293T cells using HiBiT extracellular quantification, pharmacological inhibition of lysosomal and proteasomal pathways, and BRET2 visual arrestin recruitment assay. Results The P347L mutant failed to undergo efficient outer-segment-directed trafficking and was predominantly degraded via the lysosomal pathway, consistent with its enhanced visual arrestin recruitment and endocytosis. In contrast, the L125R mutant showed protein misfolding and was degraded by both proteasomal and lysosomal pathways. In vivo, P347L mice exhibited more pronounced mitochondrial dysfunction than L125R mice, accompanied by elevated cGMP levels and lysosomal overload. Neuroinflammation was similarly present in both mutants, indicating a shared pathological mechanism rather than a differential contributor. Conclusions We propose a pathogenic model in which elevated endocytosis and mitochondrial dysfunction contribute to the accelerated photoreceptor degeneration in RHO P347L-associated adRP.
Retinitis pigmentosa (RP) is a hereditary retinal degeneration disorder often caused by mutations in the rhodopsin gene, leading to photoreceptor death and vision loss. While structural misfolding of rhodopsin is a known contributor to disease pathology, the mechanisms of its cellular and in particular metabolic consequences are poorly understood. To study the direct effects of rhodopsin misfolding and structural rescue on cellular metabolism, we used the P23A mutant and its N2C/D282C stabilized counterpart as a structural tool to assess how differences in folding stability relate to measurable changes at the metabolite level. The engineered cysteine pair allows the formation of a disulfide bond restoring structural integrity and reinforcing the stable seven-transmembrane bundle. We used untargeted Gas Chromatography-Mass Spectrometry (GC-MS) metabolomics analysis conducted in inducible rhodopsin-expressing cell lines, providing a broad and general profiling of metabolic pathway alterations in response to the expression of RP mutants and their structurally rescued counterparts. Principal component analysis, hierarchical clustering, and K-means clustering revealed distinct metabolic signatures associated with each rhodopsin-expressing cell line, demonstrating a highly significant effect of genotype on global metabolite composition (F = 71.679; R2 = 0.93724; p = 0.001). Pairwise comparisons and background-subtracted analyses identified consistent alterations in arginine and proline metabolism, glutathione metabolism, and the TCA cycle, nucleotide, amino acid metabolism, redox regulation, and mitochondrial function in cells expressing misfolded P23A. Pathway enrichment highlighted key metabolites in the respective pathways as candidate biomarkers for the rhodopsin P23A mutation. As this study employs a non-retinal cell system, the observed metabolic changes reflect conserved responses to rhodopsin misfolding and proteostatic stress in the ER rather than a direct model of rod cell degeneration. Our findings support the hypothesis that there is a biochemical link, most likely the UPR, between rhodopsin folding/misfolding status and metabolic homeostasis and suggest that targeted metabolic modulation may offer a complementary therapeutic avenue for treating RP.
M. Murthy, Hannah Staggs-Sandy, Paniz Jasbi et al.· The FASEB Journal· 0 citations
INTRODUCTION
Retinitis pigmentosa (RP) is the most common inherited neurodegenerative retinal disease. Mer receptor tyrosine kinase (MERTK) mutations are associated with severe RP and dysfunction of the RPE. Previous studies have shown that MERTK and the Rho-associated coiled-coil-containing kinases (ROCK) pathway are involved in phagocytosis. However, the specific role of the ROCK pathway in the context of MERTK-associated RP needs to be revealed.
METHODS
We established an in vitro RP cellular model via MERTK depletion in human primary retinal pigment epithelium (HsRPE) cells by siRNAs, and RCS rats with spontaneous Mertk mutations were used as RP experimental animal models. Cell viability, apoptosis, phagocytosis, and visual function were measured by MTT, TUNEL and Annexin V/propidium iodide staining, phagocytosis assays and transmission electron microscopy, and electroretinography, respectively. The expression of RhoA, ROCK, Factin, and cofilin was determined by quantitative real-time PCR, western blotting, or immunofluorescence staining.
RESULTS
MERTK knockdown substantially impaired cell survival, promoted apoptosis, and suppressed phagocytosis in HsRPE cells. In RCS rats, Mertk mutations impaired phagocytosis, promoted apoptosis of the RPE, and damaged visual functions. Silencing MERTK upregulated the phosphorylation of RhoA, ROCK2, and cofilin and decreased F-actin expression in HsRPE cells. Blocking the RhoA/ROCK axis by a selective ROCK inhibitor, Y27632, rescued the MERTK depletion-induced phagocytic dysfunction and apoptosis of HsRPE cells and rat RPE.
DISCUSSION
Our results collectively indicate that MERTK maintains RPE survival and phagocytosis via regulating the RhoA/ROCK/cofilin/F-actin axis.
CONCLUSION
This study demonstrates that MERTK deficiency impairs RPE phagocytosis and promotes cell apoptosis, leading to retinal degeneration and visual dysfunction. These findings provide new insight into RP pathogenesis and thereby offer valuable references for future drug development.
Lujia Feng, Ting Zhang, Yong Du et al.· Current Medicinal Chemistry· 0 citations
Results identify Cryaa as a critical regulator of ER stress and demonstrate that its reduction promotes UPR activation and subsequent photoreceptor apoptosis in the rd9 model, revealing a key role for Cryaa in XLRP pathology and may provide a novel therapeutic perspective for this disease.
Mingzhu Yang, Ruiqi Qiu, S. Yao et al.· Cell Death & Disease· 0 citations
Loss-of-function mutations in the ABCA4 gene cause Stargardt disease (STGD1), the most common inherited macular dystrophy leading to progressive central vision loss. Here, we generated hiPSC-derived retinal organoids harboring a premature stop codon in exon-24 of ABCA4 to evaluate the impact of this mutation on mRNA and protein levels in a human model. Immunofluorescence analysis revealed the absence of ABCA4 protein in the mutant photoreceptor outer segment discs, while single-cell RNA sequencing detected no major transcriptional alterations in rods and cones. Unexpectedly, differential gene expression and pathway enrichment analyses of Müller glial cells (MGCs) and astrocytes highlighted disruption of neuronal development, microenvironment of glial cells, intercellular communication, and programmed cell death pathways. These findings suggest that ABCA4 deficiency in photoreceptor discs may trigger early stress-associated transcriptomic responses in retinal glial cells prior to overt photoreceptor degeneration, potentially contributing to Stargardt disease pathogenesis. Human induced pluripotent stem cells (hiPSCs) were engineered to generate ABCA4-mutant cell lines, later differentiated into retinal organoids as a model of Stargardt disease. The organoids showed loss of ABCA4 from the outer segment discs of rod and cone photoreceptors, while the mutant Müller glial cells and astrocytes exhibited transcriptional changes in pathways involved in neuronal development, microenvironment, and programmed cell death. Created in https://BioRender.com.
Rossella Valenzano, A. McDonald, C. Gallego et al.· Stem Cells· 0 citations
Rhodopsin (RHO) missense variants are a leading cause of autosomal dominant retinitis pigmentosa (adRP), a progressive retinal degeneration. Interpreting RHO variant pathogenicity is challenging, and understanding their disease mechanisms is essential for developing therapeutics. We present a high-resolution map of RHO missense variant trafficking using deep mutational scanning approaches, including a surface abundance immunoassay and a complementary membrane proximity assay. This comprehensive, reproducible dataset encompassed all 6612 possible missense variants. Over 700 variants had pathogenic trafficking scores, substantially expanding the number of RHO variants with functional data. Trafficking scores correlated with the magnitude of ER stress markers and ClinVar pathogenicity classifications. Data also identified structurally clustered mutational intolerance around the intradiscal beta-plug region. Treatment with the chaperone YC-001 restored surface trafficking in most mistrafficking variants. This functional map of RHO variants provides a valuable resource for pathogenicity assessment, genotype-phenotype correlations, and the development of targeted therapeutic strategies for RHO-adRP.
K. Manian, Connor H. Ludwig, Yan Zhao et al.· Science Advances· 0 citations
Purpose Retinitis pigmentosa (RP) is a hereditary retinal disease characterized by progressive photoreceptor cell (PRC) degeneration. WD repeat domain 34 (WDR34), an intermediate chain of dynein-2, is essential for retrograde intraflagellar transport (IFT). However, the mechanisms by which WDR34 deficiency causes retinal degeneration remain unclear. This study aims to investigate the impact of WDR34 deficiency on retrograde IFT and its contribution to retinal degeneration. Methods WDR34 deficiency was modeled in vivo via subretinal injection of adeno-associated virus–shRNA–WDR34 and in vitro by CRISPR/Cas9-mediated knockout in 661W cells. Retinal degeneration and IFT defects were assessed by histologic, functional, and ultrastructural analyses. Proteomic analysis followed by in vivo validation was used to investigate the molecular mechanism underlying WDR34-deficient retinal degeneration. Results WDR34 knockdown induced progressive retinal degeneration characterized by PRC apoptosis, gradual outer nuclear layer thinning, reduced electroretinography responses, and outer segment shortening. WDR34 deficiency impaired retrograde IFT and caused rhodopsin and opsin mislocalization. These alterations induced endoplasmic reticulum stress and unfolded protein response (UPR) activation, activating the IRE1α/TRAF2/NF-κB signaling pathway, ultimately contributing to retinal inflammation and degeneration. Conclusions WDR34 is crucial for maintaining retrograde IFT in PRCs. WDR34 deficiency disrupts outer segment maintenance and triggers UPR-mediated inflammatory responses and apoptosis, ultimately leading to retinal degeneration. This study reveals a novel mechanistic link among WDR34, retrograde IFT, ciliopathies, and retinal degeneration, providing potential therapeutic insights for ciliopathy-associated RP.
Bo Jia, Jianan Xie, Xuebin Zhou et al.· Investigative Ophthalmology...· 0 citations