Preservation of Mitochondrial Function by SkQ1 in Skin Fibroblasts Derived from Patients with Leber's Hereditary Optic Neuropathy Is Associated with the PINK1/PRKN-Mediated Mitophagy.

Increased or altered mitochondrial ROS production in the retinal ganglion cells is regarded as the chief culprit of the disease-causing Leber's hereditary optic neuropathy (LHON). SkQ1 is a rechargeable mitochondria-targeted antioxidant with high specificity and efficiency. SkQ1 has already been used to treat LHON patients, and a phase 2a randomized clinical trial of SkQ1 has demonstrated improvements in eyesight. However, the underlying mechanism of SkQ1 in LHON remains unclear. This study aimed to assess the effects and molecular mechanism of SkQ1 in the preservation of mitochondrial function using skin fibroblasts derived from LHON patients. Our study found that SkQ1 could reduce ROS production and stabilize the mitochondrial membrane. Mechanistically, through network pharmacology and molecular docking, we identified the key targets of SkQ1 as SOD2 and PINK1, which play crucial roles in redox and mitophagy. SkQ1 interacted with PINK1 and downregulated its expression to balance mitochondrial homeostasis. Collectively, the findings of our study reveal that by regulating PINK1/PRKN-mediated mitophagy, SkQ1 preserves mitochondrial function in LHON fibroblasts. The data indicate that SkQ1 may be a novel therapeutic intervention to prevent the progression of LHON.

Proteomics identifies multiple retinitis pigmentosa associated proteins involved in retinal degeneration in a mouse model bearing a Pde6b mutation.

Retinitis pigmentosa (RP) is a progressive and degenerative retinal disease resulting in severe vision loss. RP have been extensively studied for pathogenetic mechanisms and treatments. Yet there is little information about alterations of RP associated proteins in phosphodiesterase 6 beta (Pde6b) mutated model. To explore the roles of RP causing proteins, we performed a label free quantitative mass spectrometry based proteomic analysis in rd10 mouse retinas. 3737 proteins were identified at the degenerative time points in rd10 mice. 222 and 289 differentially expressed proteins (DEPs) (fold change, FC > 2, p < 0.05) were detected at 5 and 8 weeks. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, visual perception and phototransduction were severely affected. The downregulated DEPs were significantly enriched in cilium assembly and protein localization. 25 decreased DEPs causing autosomal recessive/dominant retinitis pigmentosa were visualized by heatmaps. Protein-protein interaction network represented 13 DEPs interacted directly with Pde6b protein. 25 DEPs causing RP were involved in phototransduction, visual perception, response to stimulus, protein localization and cilium assembly pathways. The significantly reduced expressions of DEPs were further validated by quantitative reverse transcription polymerase chain reaction (qPCR), Western blots (WB) and immunohistochemistry (IHC). This study revealed the molecular mechanisms underlying early and late stage of RP, as well as changes of RP-causing proteins.

Retinal Proteomic Alterations and Combined Transcriptomic-Proteomic Analysis in the Early Stages of Progression of a Mouse Model of X-Linked Retinoschisis.

X-linked retinoschisis (XLRS) is among the most commonly inherited degenerative retinopathies. XLRS is caused by functional impairment of RS1. However, the molecular mechanisms underlying RS1 malfunction remain largely uncharacterized. Here, we performed a data-independent acquisition-mass spectrometry-based proteomic analysis in RS1-null mouse retina with different postal days (Ps), including the onset (P15) and early progression stage (P56). Gene set enrichment analysis showed that type I interferon-mediated signaling was upregulated and photoreceptor proteins responsible for detection of light stimuli were downregulated at P15. Positive regulation of Tor signaling was downregulated and nuclear transcribed mRNA catabolic process nonsense-mediated decay was upregulated at P56. Moreover, the differentially expressed proteins at P15 were enriched in metabolism of RNA and RNA destabilization. A broader subcellular localization distribution and enriched proteins in visual perception and phototransduction were evident at P56. Combined transcriptomic-proteomic analysis revealed that functional impairments, including detection of visible light, visual perception, and visual phototransduction, occurred at P21 and continued until P56. Our work provides insights into the molecular mechanisms underlying the onset and progression of an XLRS mouse model during the early stages, thus enhancing the understanding of the mechanism of XLRS.

Identification of Age-associated Proteins and Functional Alterations in Human Retinal Pigment Epithelium.

Retinal pigment epithelium (RPE) has essential functions, such as nourishing and supporting the neural retina, and is of vital importance in the pathogenesis of age-related retinal degeneration. However, the exact molecular changes of RPE during aging remain poorly understood. Here, we isolated human primary RPE (hRPE) cells from 18 eye donors distributed over a wide age range (10-67 years old). A quantitative proteomic analysis was performed to analyze changes in their intracellular and secreted proteins. Age-group related subtypes and age-associated proteins were revealed and potential age-associated mechanisms were validated in ARPE-19 and hRPE cells. The results of proteomic data analysis and verifications suggest that RNF123- and RNF149-related protein ubiquitination plays an important role in protecting hRPE cells from oxidative damage during aging. In older hRPE cells, apoptotic signaling-related pathways were up-regulated, and endoplasmic reticulum organization was down-regulated both in the intracellular and secreted proteomes. Our work paints a detailed molecular picture of hRPE cells during the aging process and provides new insights into the molecular characteristics of RPE during aging and under other related clinical retinal conditions.

The Protective Effects of VVN001 on LPS-Induced Inflammatory Responses in Human RPE Cells and in a Mouse Model of EIU.

To investigate protective effects of VVN001 on lipopolysaccharide (LPS)-induced inflammatory response in human retinal pigment epithelial (RPE) cells and in a mouse model of endotoxin-induced uveitis (EIU), and to explore the underlying mechanisms. Human primary RPE (hRPE) and ARPE-19 cells were pretreated with or without VVN001 for 1 h followed by 10 µg/mL LPS stimulation for 24 h. mRNA, and protein levels of inflammatory cytokines were analyzed with real-time PCR, western blotting, and ELISA. EIU was induced by intravitreal injection of 125 ng LPS in female BALB/c mice. VVN001 eye drops (1%) were locally administrated every 4 h for 24 h after LPS injection. Clinical scores were assessed with a slit lamp. mRNA and protein levels of inflammatory cytokines were investigated simultaneously. Compared with the LPS group, VVN001 pretreatment significantly reduced mRNA expressions of intercellular adhesion molecule-1 (ICAM-1), IL-6, IL-8, TNF-α, IL-1ß, IL-18, caspase-1 in hRPE, and ARPE-19 cells. Protein overproduction of ICAM-1, TNF-α, IL-1ß, NLRP3, caspase-1 P20, and p-IκBα/IκBα stimulated by LPS was suppressed by VVN001 pretreatment. In vivo, VVN001 significantly reduced the average clinical score from 5.0 to 1.3 in EIU mice. Furthermore, overproduction of ICAM-1, IL-1ß, NLRP3, caspase-1 P20, and p-IκBα/IκBα at mRNA and protein levels were remarkably suppressed by VVN001. VVN001 alleviated the inflammatory response induced by LPS both in vitro and in vivo. The effect of anti-inflammation is associated with inhibiting the overproduction of ICAM-1 and blocking the activation of NLRP3 inflammasome and the NF-κB signaling pathway.

Intravitreal Injection of an Exosome-Associated Adeno-Associated Viral Vector Enhances Retinoschisin 1 Gene Transduction in the Mouse Retina.

To investigate whether exosome-associated adeno-associated virus (AAV) retinoschisin 1 (RS1) vector improved the transduction efficiency of RS1 in the mouse retina. pAAV2-RS1-ZsGreen plasmid was constructed by homologous recombination. Exosome-associated AAV vectors containing human RS1 gene (exosome-associated AAV [exo-AAV]2-RS1-ZsGreen) were isolated from producer cells' supernatant, and confirmed by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. In vitro, HEK-293T cells were transduced with AAV2-RS1-ZsGreen and exo-AAV2-RS1-ZsGreen. In vivo, 1 µL of AAV2-RS1-ZsGreen or 1 µL exo-AAV2-RS1-ZsGreen (2 × 108 genome copies/µL) was injected intravitreally into the C57BL/6J mouse eyes. Phosphate buffer saline was injected as controls. The mRNA and the protein expression in the retina were detected. Exo-AAV2-RS1-ZsGreen possessed lipid bilayers, a saucer-like structures and an average of 120 nm particle size. The expression of RS1 and ZsGreen in exo-AAV2-RS1-ZsGreen group were 7.6 times and 5.7 times that of AAV2-RS1-ZsGreen group in HEK-293T cells, respectively. Furthermore, RS1 protein expression increased by 11.8 times in HEK-293T cells. Intravitreal injection of exo-AAV significantly increased the transduction efficiency of RS1 than AAV. Exo-AAV may be a powerful gene delivery system for gene therapy of X-link retinoschisis as well as other inherited retina degenerations.

P2X7 Receptor Antagonist Attenuates Retinal Inflammation and Neovascularization Induced by Oxidized Low-Density Lipoprotein.

Age-related macular degeneration (AMD) is a common and severe blinding disease among people worldwide. Retinal inflammation and neovascularization are two fundamental pathological processes in AMD. Recent studies showed that P2X7 receptor was closely involved in the inflammatory response. Here, we aim to investigate whether A740003, a P2X7 receptor antagonist, could prevent retinal inflammation and neovascularization induced by oxidized low-density lipoprotein (ox-LDL) and explore the underlying mechanisms. ARPE-19 cells and C57BL/6 mice were treated with ox-LDL and A740003 successively for in vitro and in vivo studies. In this research, we found that A740003 suppressed reactive oxygen species (ROS) generation and inhibited the activation of Nod-like receptor pyrin-domain protein 3 (NLRP3) inflammasome and nuclear factor-κB (NF-κB) pathway. A740003 also inhibited the generation of angiogenic factors in ARPE-19 cells and angiogenesis in mice. The inflammatory cytokines and phosphorylation of inhibitor of nuclear factor-κB alpha (IKBα) were repressed by A740003. Besides, ERG assessment showed that retinal functions were remarkably preserved in A740003-treated mice. In summary, our results revealed that the P2X7 receptor antagonist reduced retinal inflammation and neovascularization and protected retinal function. The protective effects were associated with regulation of NLRP3 inflammasome and the NF-κB pathway, as well as inhibition of angiogenic factors.

A Novel Enzyme-Free Biosensor Based on Porous Core-Shell Metal Organic Frame Nanocomposites Modified Electrode for Highly Sensitive Detection of Uric Acid and Dopamine.

A novel enzyme-free biosensor based on porous metal organic frame nanocomposites, i.e., core-shell structured Au@NC@GC nanocomposites, has been constructed for simultaneous determination of uric acid (UA) and dopamine (DA). Au@ZIF-8@ZIF-67 was prepared through a seed-mediated growth method and carbonized in nitrogen atmosphere to synthesize a nanoporous hybrid carbon materials (Au@NC@GC). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) studies demonstrate that the as-prepared Au@NC@GC modified glassy carbon electrode (Au@NC@GC-GCE) possesses a high selectivity and sensitivity for simultaneous detections of UA and DA. It exhibited wide linear responses for UA and DA in the range from 10 µM to 600 µM and 10 µM to 150 µM, with the detection limits of 0.773 nM and 0.746 nM, respectively (S/N = 3). Moreover, this novel electrochemical biosensor could be further utilized in biological analysis (i.e., human serum), and the satisfactory recovery results of UA and DA could be readily obtained. These afore-mentioned results further manifest that the as-prepared biosensors are capable for quantitatively monitoring UA and DA in serum, verifying the possibility for its future promising applications in real biological or clinic samples analysis.

AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis.

JOURNAL/nrgr/04.03/01300535-202508000-00030/figure1/v/2024-09-30T120553Z/r/image-tiff Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death. However, there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation. Adeno-associated virus (AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa. The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function. To do this, we injected retinal degeneration 10 (rd10) mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark- and light-adapted electroretinogram, optical coherence tomography, and immunofluorescence. Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment, and the results from this analysis were verified by real-time polymerase chain reaction and western blotting. AAV2-PDE6B injection significantly upregulated PDE6ß expression, preserved electroretinogram responses, and preserved outer nuclear layer thickness in rd10 mice. Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception, and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice. Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways. Furthermore, the phototransduction-related proteins Pde6α, Rom1, Rho, Aldh1a1, and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment. Finally, Bax/Bcl-2, p-ERK/ERK, and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment. Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.