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.
Abstract
X-linked Retinitis Pigmentosa (XLRP) is an inherited retinopathy predominantly caused by
RPGR
mutations, ultimately leading to photoreceptor apoptosis and vision loss. Although the genetic basis is well established, the downstream pathogenic and molecular mechanisms remain inadequately elucidated. The aim of this study is to explore the pathogenesis from the perspective of endoplasmic reticulum (ER) stress, which was previously underexplored. Using
rd9
mouse retinas and mouse embryonic fibroblast (MEF) cells, we confirmed the presence of ER stress, activation of the unfolded protein response (UPR), and photoreceptor apoptosis through Western blot and immunofluorescence (IF) analyses. Furthermore, knockdown of Cryaa in 661W cells directly induced ER stress and UPR activation. Transmission electron microscopy (TEM) photomicrographs revealed significant ultrastructural alterations in ER and mitochondria within
rd9
retinas and MEF cells. Proteomic analyses of
rd9
retinas identified significantly enriched pathways including UPR, protein folding, and unfolded protein binding, associated with Cryaa downregulation. Collectively, our 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. These findings reveal, for the first time, a key role for Cryaa in XLRP pathology and may provide a novel therapeutic perspective for this disease.
Ribosomal L1 domain-containing protein 1 (RSL1D1) is an RNA-binding protein that relates to senescence. Nevertheless, the mechanism of RSL1D1 in modulating senescence and ferroptosis in diabetic retinopathy (DR) remains undefined. The DR mice were developed by intraperitoneal injection of STZ, and AAV targeting RPE was used for gene intervention. ARPE-19 cells were infected using oe-RSL1D1 and sh-FTH1 lentivirus before 30 mM high glucose (HG) exposure. Cell damage was assessed by measuring the expression of senescence and ferroptosis markers in ARPE-19 cells, along with oxidative stress indicators and Fe2+ levels. RSL1D1 and FTH1 expression were significantly reduced in the retinal pigment epithelium (RPE) layer of diabetic mice and HG-induced ARPE-19 cells. Downregulation of RSL1D1 led to RPE cell senescence and dysregulated iron homeostasis-induced ferroptosis. HG treatment reduced the interaction between RSL1D1 protein and FTH1 mRNA in ARPE-19 cells, while RSL1D1 overexpression enhanced this interaction, thereby stabilizing FTH1 mRNA expression. The alleviating effects of RSL1D1 overexpression on retinal pigment epithelium cell senescence and ferroptosis in vitro and in vivo were compromised by FTH1 knockdown. Overall, this study unveils a posttranscriptional regulation of FTH1 by RSL1D1 and uncovers the implication of RSL1D1/FTH1 in cellular senescence and ferroptosis in DR.
L. Qin, Yanli Guo, Yi Zhang et al.· Biochimica et biophysica act...· 0 citations
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.
Retinal degenerative diseases are a leading cause of irreversible blindness. Their pathogenesis is intricately linked to oxidative stress-induced dysfunction of retinal pigment epithelial (RPE) cells and subsequent retinal degeneration. Macroautophagy/autophagy, a critical cellular degradation pathway, plays a vital role in maintaining RPE homeostasis, yet its dysregulation in retinal degenerative diseases remains poorly understood. In this study, we observed that sodium iodate (NaIO3), an oxidative stress inducer, triggered lysosomal dysfunction via lysosomal membrane permeabilization (LMP), thereby impairing autophagic flux in RPE cells and exacerbating retinal degeneration. RNA sequencing identified Lamp3 (lysosomal-associated membrane protein 3) as a downregulated gene following NaIO3 treatment. Functionally, LAMP3 overexpression alleviated NaIO3-induced LMP, improved lysosomal function, and alleviated autophagic impairment. Furthermore, upregulation of LAMP3 reduced oxidative stress and apoptosis in RPE cells, while alleviating retinal degeneration in a NaIO3-induced mouse model. Mechanistically, our data suggested that NaIO3 upregulated the transcription factor SNAI1, which acts as a transcriptional repressor of LAMP3. SNAI1 knockdown increased LAMP3 expression, thereby facilitating the recovery of lysosomal function and the alleviation of autophagic impairment. Collectively, our findings indicate that the SNAI1-LAMP3 axis contributes to the regulation of the autophagy-lysosomal pathway in retinal degeneration, highlighting a potential therapeutic target for delaying disease progression.
Yuke Ji, Yanan Sun, Xiaosheng Huang et al.· Autophagy· 0 citations
BACKGROUND
Diabetic retinopathy (DR) is one of the most prevalent complications of diabetes which could lead to vision impairment. Dysfunction of the retinal pigment epithelium (RPE) is an early pathogenic event, where various mechanisms may contribute to the disease progression. circular RNAs (circRNAs) have been reported to be important regulators in diabetic complications, acting as miRNA sponge, participating in the regulation of gene transcription or coding short peptides.
METHODS
Circular RNA microarray and RNA-seq were used to identify differentially expressed circRNAs in ARPE-19 cells in normal and high glucose treatments. The expression of cKIAA1462, miR-183-5p, and high mobility group box 1 (HMGB1) were determined using quantitative real-time polymerase chain reaction and Western blot. In vitro experiments, such as flow cytometry, Western blot, and Electron microscope (TEM) were conducted. The binding interaction was confirmed using dual-luciferase reporter and overexpression/inhibition experiments. cKIAA1462 was knocked down via intravitreal lentiviral injection in diabetic mice, followed by expression level detection, functional analysis and histological assessments.
RESULTS
cKIAA1462 was significantly upregulated under high glucose conditions both in vitro and in vivo (in retinas of diabetic mice). It acted as a molecular sponge for miR-183-5p, increasing the expression of HMGB1. Elevated HMGB1 concurrently impaired autophagic flux (increased p62, decreased autophagosomes) and activated the NLRP3 inflammasome (upregulated NLRP3, ASC, caspase-1), promoting pyroptosis. Silencing cKIAA1462 in vivo restored autophagy, suppressed pyroptosis, improved retinal structure, and enhanced electroretinogram responses in diabetic mice.
CONCLUSION
The cKIAA1462/miR-183-5p/HMGB1 axis plays a critical role in diabetic RPE injury by dual regulation of autophagy and pyroptosis. Targeting this pathway may offer a novel therapeutic strategy for early diabetic retinopathy.
Lufei Wang, Jia’nan Xie, Longfei Yang et al.· Journal of Translational Med...· 0 citations
Retinitis pigmentosa (RP) is the most common inherited retinal degenerative disease leading to blindness. RP is characterized by progressive loss of photoreceptors and retinal pigment epithelium (RPE), leading to retinal degeneration. The mechanisms that initiate RP and drive retinal vulnerability are poorly understood, and new strategies for preventing and treating RP are urgently needed. Although mitochondrial dysfunction initiates many neurodegenerative diseases, the contribution of mitochondrial dysfunction to RP is unclear. Single-cell RNA sequencing, transmission electron microscopy, and enzyme-linked immunosorbent assays revealed that photoreceptor and RPE cells have abnormal mitochondria in rats with RP. Nicotinamide adenine dinucleotide (NAD+) metabolism decreased in rats with RP, increasing the vulnerability to disease-related insults. Similar experimental results were observed in a Mer tyrosine kinase receptor (MERTK)-associated RP primary human RPE cell model. Electroretinography, immunofluorescence, and fundus photography revealed that oral administration of the NAD+ precursor nicotinamide mononucleotide (NMN) protected rats with RP from retinal degeneration. Single-cell RNA sequencing, siRNA targeting, and Adeno-associated virus applications demonstrated that NMN elicits therapeutic effects via the glyceraldehyde-phosphate dehydrogenase-mitochondria pathway. These results indicate that mitochondrial abnormalities may be drivers of RP, and NMN elicits therapeutic effects on RP. Retinitis pigmentosa leads to blindness due to photoreceptor loss. Here, the authors show that the NAD+ precursor NMN protects against retinal degeneration by improving mitochondrial function in MERTK-associated models, offering potential therapeutic insights.
AIM: To investigate the key role of the mitochondrial outer membrane protein FUN14 domain-containing 2 (FUNDC2) in retinal pigment epithelium (RPE) ferroptosis during retinitis pigmentosa (RP) progression.
METHODS: Unbiased label-free proteomics was employed to identify differentially expressed proteins in the RPE of a sodium iodate (SI)-induced rat model. In vitro experiments were conducted using human retinal pigment epithelial (ARPE)-19 cells. The effects of SI treatment and FUNDC2 knockdown on cell viability and the expression of ferroptosis-protective molecules, including glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), ferritin heavy chain 1 (FTH1), and solute carrier family 25 member 11 (SLC25A11) were evaluated.
RESULTS: Proteomic analysis revealed that FUNDC2 was significantly upregulated in the RPE of SI-induced rats. In ARPE-19 cells, SI treatment significantly increased FUNDC2 expression while decreasing the levels of ferroptosis-protective molecules. Functional experiments demonstrated that knocking down FUNDC2 effectively rescued SI-induced loss of cell viability and restored GPX4 expression.
CONCLUSION: These findings provide the first evidence that FUNDC2 acts as a potential upstream regulator of RPE ferroptosis in RP, at least partially by negatively regulating GPX4. Consequently, FUNDC2 is a potential therapeutic target for the future treatment of RP.
Jie-yu Chen, Yu Hong· International Journal of Oph...· 0 citations