Oxidative stress represents one of the principal inductors of lifestyle-related and genetic diseases. Among them, inherited retinal dystrophies, such as age-related macular degeneration and retinitis pigmentosa, are well known to be susceptible to oxidative stress. To better understand how high reactive oxygen species levels may be involved in retinal dystrophies onset and progression, we performed a whole RNA-Seq experiment. It consisted of a comparison of transcriptomes’ profiles among human retinal pigment epithelium cells exposed to the oxidant agent N-retinylidene-N-retinylethanolamine (A2E), considering two time points (3h and 6h) after the basal one. The treatment with A2E determined relevant differences in gene expression and splicing events, involving several new pathways probably related to retinal degeneration. We found 10 different clusters of pathways involving differentially expressed and differentially alternative spliced genes and highlighted the sub- pathways which could depict a more detailed scenario determined by the oxidative-stress-induced condition. In particular, regulation and/or alterations of angiogenesis, extracellular matrix integrity, isoprenoid-mediated reactions, physiological or pathological autophagy, cell-death induction and retinal cell rescue represented the most dysregulated pathways. Our results could represent an important step towards discovery of unclear molecular mechanisms linking oxidative stress and etiopathogenesis of retinal dystrophies.
Inherited retinal dystrophies are characterized by photoreceptor death. Oxidative stress usually occurs, increasing vision loss, and oxidative damage is often reported in retinitis pigmentosa (RP). More than 300 genes have been reported as RP causing. In contrast, choroidal neovascularization (CNV) only occasionally develops in the late stages of RP. We herein study the regulation of RP causative genes that are likely linked to CNV onset under oxidative conditions. We studied how the endogenous adduct N-retinylidene-N-retinylethanolamine (A2E) affects the expression of angiogenic markers in human retinal pigment epithelium (H-RPE) cells and a possible correlation with RP-causing genes. H-RPE cells were exposed to A2E and blue light for 3 and 6h. By transcriptome analysis, genes differentially expressed between A2E-treated cells and untreated ones were detected. The quantification of differential gene expression was performed by the Limma R package. Enrichment pathway analysis by the FunRich tool and gene prioritization by ToppGene allowed us to identify dysregulated genes involved in angiogenesis and linked to RP development. Two RP causative genes, AHR and ROM1, can be associated with an increased risk of CNV development. Genetic analysis of RP patients affected by CNV will confirm this hypothesis.
Deep analysis of regulative mechanisms of transcription and translation in eukaryotes could improve knowledge of many genetic pathologies such as retinitis pigmentosa ( RP ). New layers of complexity have recently emerged with the discovery that ‘junk’ DNA is transcribed and, among these, miRNAs have assumed a preponderant role. We compared changes in the expression of mi RNA s obtained from whole transcriptome analyses, between two groups of retinal pigment epithelium ( RPE ) cells, one untreated and the other exposed to the oxidant agent oxidized low‐density lipoprotein (ox LDL ), examining four time points (1, 2, 4 and 6 h). We found that 23 mi RNA s exhibited altered expression in the treated samples, targeting genes involved in several biochemical pathways, many of them associated to RP for the first time, such as those mediated by insulin receptor signaling and son of sevenless. Moreover, five RP causative genes ( KLHL7 , RDH11 , CERKL , AIPL1 and USH1G ) emerged as already validated targets of five altered mi RNA s (hsa‐miR‐1307, hsa‐miR‐3064, hsa‐miR‐4709, hsa‐miR‐3615 and hsa‐miR‐637), suggesting a tight connection between induced oxidative stress and RP development and progression. This mi RNA expression analysis of oxidative stress‐induced RPE cells has discovered new regulative functions of mi RNA s in RP that should lead to the discovery of new ways to regulate the etiopathogenesis of RP .
Long non-coding RNAs (lncRNAs) are untranslated transcripts which regulate many biological processes. Changes in lncRNA expression pattern are well-known related to various human disorders, such as ocular diseases. Among them, retinitis pigmentosa, one of the most heterogeneous inherited disorder, is strictly related to oxidative stress. However, little is known about regulative aspects able to link oxidative stress to etiopathogenesis of retinitis. Thus, we realized a total RNA-Seq experiment, analyzing human retinal pigment epithelium cells treated by the oxidant agent N-retinylidene-N-retinylethanolamine (A2E), considering three independent experimental groups (untreated control cells, cells treated for 3 h and cells treated for 6 h). Differentially expressed lncRNAs were filtered out, explored with specific tools and databases, and finally subjected to pathway analysis. We detected 3,3'-overlapping ncRNAs, 107 antisense, 24 sense-intronic, four sense-overlapping and 227 lincRNAs very differentially expressed throughout all considered time points. Analyzed lncRNAs could be involved in several biochemical pathways related to compromised response to oxidative stress, carbohydrate and lipid metabolism impairment, melanin biosynthetic process alteration, deficiency in cellular response to amino acid starvation, unbalanced regulation of cofactor metabolic process, all leading to retinal cell death. The explored lncRNAs could play a relevant role in retinitis pigmentosa etiopathogenesis, and seem to be the ideal candidate for novel molecular markers and therapeutic strategies.
Mitochondria are subject to continuous oxidative stress stimuli that, over time, can impair their genome and lead to several pathologies, like retinal degenerations. Our main purpose was the identification of mtDNA variants that might be induced by intense oxidative stress determined by N-retinylidene-N-retinylethanolamine (A2E), together with molecular pathways involving the genes carrying them, possibly linked to retinal degeneration. We performed a variant analysis comparison between transcriptome profiles of human retinal pigment epithelial (RPE) cells exposed to A2E and untreated ones, hypothesizing that it might act as a mutagenic compound towards mtDNA. To optimize analysis, we proposed an integrated approach that foresaw the complementary use of the most recent algorithms applied to mtDNA data, characterized by a mixed output coming from several tools and databases. An increased number of variants emerged following treatment. Variants mainly occurred within mtDNA coding sequences, corresponding with either the polypeptide-encoding genes or the RNA. Time-dependent impairments foresaw the involvement of all oxidative phosphorylation complexes, suggesting a serious damage to adenosine triphosphate (ATP) biosynthesis, that can result in cell death. The obtained results could be incorporated into clinical diagnostic settings, as they are hypothesized to modulate the phenotypic expression of mtDNA pathogenic variants, drastically improving the field of precision molecular medicine.
The neurovascular unit (NVU) is a relatively recent concept that clearly describes the relationship between brain cells and their blood vessels. The components of the NVU, comprising different types of cells, are so interrelated and associated with each other that they are considered as a single functioning unit. For this reason, even slight disturbances in the NVU could severely affect brain homeostasis and health. In this review, we aim to describe the current state of knowledge concerning the role of oxidative stress on the neurovascular unit and the role of a single cell type in the NVU crosstalk.
A highly reduced activity of the ELOVL4 promoter was registered due to combination of two variants. Decrease of ELOVL4 enzymatic activity could lead to a deficiency of VLC-PUFA, essential components for rods function and longevity, which are among the parameters involved in the etiopathogenesis of STGD.
Glyoxalase 1 (GLO1) is a ubiquitous cellular enzyme involved in detoxification of methylglyoxal (MGO), a cytotoxic byproduct of glycolysis, whose excess can cause oxidative stress. In retinitis pigmentosa (RP), the prevalent cause of blindness just during working life in the industrialized countries, oxidative stress represents one of the possible mechanisms leading to death of cones following that of rods in the retina. To date, the causes of secondary death of cones remain unclear and among proposed mechanisms are: the deprivation of trophic factors normally produced by healthy rods, a compromised uptake of nutrients to cones due to irreversible destruction of RPE-cone outer segment, microglial activation and following release of pro-inflammatory cytokines and rod-derived toxins. In present paper, role of oxidative stress due to an excess of MGO was evaluated. In particular, we wanted to determine whether single nucleotide polymorphisms (SNPs) in GLO1 influence enzyme activity, contributing to cone death in advanced RP. 120 healthy controls and 80 RP patients from Sicilian population were genotyped for three GLO1 common SNPs, rs1130534 (c.372A>T, p.G124G), rs2736654 (c.A332C, p.E111A) and rs1049346 (c.-7C>T, 5'-UTR). While c.A332C polymorphism was not associated with RP, c.372A>T showed an allelic association (T372 allele frequency = 70% vs 60% in controls, p = 0.0071). Conversely, c.-7C>T showed both genotypic (χ = 68.0952; p = 1.634e-15) and allelic associations (χ = 51.7094; p = 6.435e-13): mutated allele frequency was higher in controls than in patients, suggesting its possible protective role. RP susceptibility may be associated with two of the analyzed GLO1 polymorphisms (rs1130534 and rs1049346).
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