Biology subjects

Armento, A.

Publications and source records attributed to Armento, A..

8 recordsLinked to original sources

bioRxiv2026

A mutation-agnostic and allele-specific ASO strategy demonstrates potent functional rescue and retinal preservation in RHO-linked retinitis pigmentosa

Autosomal dominant retinitis pigmentosa (adRP) caused by RHO mutations is a leading form of inherited retinal degeneration. Extensive allelic heterogeneity of RHO pathogenic variants limits the translational applicability of mutation-specific gene therapies. To address this, we developed SNARE (SNP-guided Silencing of Aberrant RHO Expression), a mutation-independent, allele-specific antisense oligonucleotide (ASO) strategy. SNARE selectively suppresses mutant RHO transcripts by targeting the common, benign c.-26A/G single-nucleotide polymorphism (SNP) as an allelic discriminator. Candidate gapmer ASOs were screened in engineered reporter lines and validated in patient-derived retinal organoids, identifying RHOligo-A as the lead c.-26A-targeting candidate. In vitro, RHOligo-A achieved robust, preferential knockdown of the target allele, improving RHO localization in retinal organoids, and demonstrated a favorable safety profile with minimal transcriptomic off-target effects and no detectable immunostimulatory activity. Subsequent validation in a novel, humanized RHOP347L/WT mouse model, achieved sustained c.-26A-linked allele-selective suppression, retinal structure preservation, and significantly restored visual function, upon a single intravitreal administration. These findings establish RHOligo-A and SNARE as a scalable, mutation-independent therapeutic platform with strong translational potential and substantial clinical reach for RHO-associated adRP.

genetics↗

bioRxiv2026

VCP inhibition preserves photoreceptor integrity under hydroquinone-induced oxidative stress in a human iPSC-RPE/porcine neuroretina co-culture model

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in older adults. It is characterised by early retinal pigment epithelium (RPE) dysfunction followed by progressive photoreceptor degeneration. Cigarette smoking is a major environmental risk factor for AMD, and hydroquinone (HQ), a redox-active cigarette smoke component, induces oxidative stress and apoptosis in RPE cells. To analyse how RPE stress contributes to photoreceptor degeneration, we employed a retinal co-culture model composed of human induced pluripotent stem cell-derived RPE (iPSC-RPE) cells in conjunction with porcine neuroretina explants. Exposure to HQ induced oxidative stress in iPSC-RPE cells as well as retinal photoreceptors (RPR), resulting in apoptosis, executed at least in part by caspase activation. Concomitantly, HQ caused endoplasmic reticulum (ER) stress (ERAD) in RPR followed by their degeneration, evidenced by reduced outer nuclear layer (ONL) rows and shortened RPR outer segments (OS). Based on earlier results, which suggest a perturbation of proteostasis due to HQ, we tested whether ML240, a bona fide inhibitor of valosin-containing protein (VCP), would influence the degree of degenerative activities. ML240 did not prevent HQ-induced apoptosis in iPSC-RPE cells. However, it significantly preserved photoreceptor integrity, retaining OS length and cone density in HQ-stressed co-cultures. Proteomic analysis suggested that ML240 reshapes stress response patterns of the HQ-exposed neuroretina, as evidenced by a reduction in ERAD-associated markers, increased levels of antioxidant response proteins, and the preservation of cytochrome c enrichment in photoreceptor inner segments, which indicates improved mitochondrial integrity consistent with the observed preservation of photoreceptor structure. Together, these findings establish the iPSC-RPE/neuroretina co-culture as a platform to analyse pathophysiological features of AMD, dissect cell type-specific retinal responses to environmental stress and test neuroprotective pharmacological approaches to protect photoreceptors in oxidative stress-associated retinal degeneration.

cell biology↗

bioRxiv2024

The AMD-associated genetic polymorphism CFH Y402H confers vulnerability to Hydroquinone-induced stress in iPSC-RPE cells

Age-related macular degeneration (AMD), a degenerative disease of the macula, is caused by an interplay of diverse risk factors (genetic predisposition, age and lifestyle habits). One of the main genetic risks includes the Y402H polymorphism in complement Factor H (FH), an inhibitor of complement system activation. There has been, and continues to be, much discussion around the functional consequences of this Y402H polymorphism, whether the soluble FH protein confers its risk association, or if the cells expressing the protein themselves are affected by the genetic alteration. In our study, we examined the cell characteristics of the retinal pigment epithelium (RPE) cells, which play a major role in retinal homeostasis and stability and which are synonymously linked to AMD. Here, we employ RPE cells derived from induced pluripotent stem cells (iPSC) generated from donors, carrying either homozygous 402Y (low risk) or 402H (high risk) variants of the CFH gene. RPE cells were treated with Hydroquinone (HQ), a component of cigarette smoke, to induce oxidative damage. Intriguingly, RPE cells carrying high genetic risk proved more vulnerable to oxidative insult when exposed to HQ, as demonstrated by increased cytotoxicity, caspase activation, compared to the low-risk RPE cells. The exposure of RPE cells to RPE conditioned medium, normal human serum (NHS) and inactivated NHS (iNHS) had minimal impact on cell cytotoxicity and caspase activation, nor did the presence of purified soluble FH rescue the observed effects. This suggests that the degree of cellular susceptibility to oxidative stress is not conferred by soluble FH protein and other complement sources, but intercellularly because of the corresponding genetic risk predisposition. Considering the known connection of oxidative stress to proteotoxic stress and degrading processes, we investigated the unfolded protein response (UPR) and autophagy. When exposed to HQ, RPE cells showed an increase in autophagy markers, however iPSC-RPE cells carrying high genetic risk showed an overall reduced autophagic flux. Our data support the hypothesis that RPE cells carrying high genetic risk are less resilient to oxidative stress.

cell biology↗

bioRxiv2021

mTOR inhibition via Rapamycin treatment partially reverts the deficit in energy metabolism caused by FH loss in RPE cells

Age-related macular degeneration (AMD) is a complex degenerative disease of the retina with multiple risk-modifying factors, including ageing, genetics and lifestyle choices. The combination of these factors leads to oxidative stress, inflammation and metabolic failure in the retinal pigment epithelium (RPE) with subsequent degeneration of photoreceptors in the retina. The alternative complement pathway is tightly linked to AMD. In particular, the genetic variant in the complement factor H gene (CFH), that leads to the Y402H polymorphism in the factor H protein (FH), confers the second highest risk for the development and progression of AMD. While the association between the FH Y402H variant and increased complement system activation is known, recent studies have uncovered novel FH functions not tied to this activity and highlighted functional relevance for intracellular FH. In our previous studies, we show that loss of CFH expression in RPE cells causes profound disturbances in cellular metabolism, increases the vulnerability towards oxidative stress and modulates the activation of pro-inflammatory signaling pathways, most importantly the NF-kB pathway. Here, we silenced CFH in hTERT-RPE1 cells to investigate the mechanism by which intracellular FH regulates RPE cell homeostasis. We found that silencing of CFH results in hyperactivation of mTOR signaling along with decreased mitochondrial respiration and that mTOR inhibition via rapamycin can partially rescue these metabolic defects. To obtain mechanistic insight into the function of intracellular FH in hTERT-RPE1 cells, we analyzed the interactome of FH via immunoprecipitation followed by Mass spectrometry-based analysis. We found that FH interacts with essential components of the ubiquitin-proteasomal pathway (UPS) as well as with factors associated with RB1/E2F signalling in a complement-pathway independent manner. Moreover, we found, that FH silencing affects mRNA levels of the E3 Ubiquitin-Protein Ligase Parkin and PTEN induced putative kinase (Pink1), both of them are associated with UPS. As inhibition of mTORC1 has been previoulsy shown to result in increased overall protein degradation via UPS and as FH mRNA and protein levels were shown to be affected by inhibition of UPS, our data stress a potential regulatory link between endogenous FH activity and the UPS.

molecular biology↗

bioRxiv2021

FH loss in RPE cells causes retinal degeneration in a human RPE-porcine retinal explant co-culture model

Age-related Macular degeneration (AMD) is a degenerative disease of the macula affecting the elderly population. Treatment options are limited, partly due to the lack of understanding of AMD pathology and the sparse availability of research models, that replicate the complexity of the human macula and the intricate interplay of the genetic, aging and life-style risk factors contributing to AMD. One of the main genetic risks associated with AMD is located on Complement Factor H (CFH) gene, leading to an amino acid substitution in the FH protein (Y402H). However, the mechanism of how this FH variant promotes the onset of AMD remains unclear. Previously, we have shown that FH deprivation in RPE cells, via CFH silencing, leads to increased inflammation, metabolic impairment and vulnerability towards oxidative stress. In this study, we established a novel co-culture model comprised of CFH silenced RPE cells and porcine retinal explants derived from the visual streak of the porcine eyes, closely resembling the human macula. We show that retinae exposed to FH-deprived RPE cells show signs of retinal degeneration, with rod cells being the first cells to undergo degeneration. Moreover, via Raman analyses, we observe that the main changes involve the mitochondria and lipid composition of the co-cultured retinae upon FH loss. Interestingly, the detrimental effects of FH loss in RPE cells on the neuroretina were independent of glial cell activation and external complement sources. Moreover, we show that the co-culture model is also suitable for human retinal explants, and we observed a similar trend when RPE cells deprived of FH were co-cultured with human retinal explants from a single donor eye. Our findings highlight the importance of RPE derived FH for retinal homeostasis and provide a valuable model for AMD research.

cell biology↗

bioRxiv2021

CFH loss in human RPE cells leads to inflammation and complement system dysregulation via the NF-B pathway

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease and chronic inflammatory processes may be involved. Besides ageing and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism by which CFH dysregulation confers such a great risk for AMD and its role in RPE cells homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g. IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g. C3, CFB upregulation and C5 downregulation) that are known to play a role in AMD. Moreover, we identified the NF-{kappa}B pathway as the major pathway involved in the regulation of these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-{kappa}B pathway work in synergy to maintain inflammatory and complement balance and in case either one of them is dysregulated, the RPE microenvironment changes towards a pro-inflammatory AMD-like phenotype.

immunology↗

bioRxiv2020

Loss of Complement Factor H impairs antioxidant capacity and energy metabolism of human RPE cells

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly population. About 50% of AMD patients present polymorphisms in the Complement Factor H (CFH) gene, coding for Factor H protein (FH). AMD-associated CFH risk variants, Y402H in particular, impair FH function leading to complement overactivation. In AMD, retinal homeostasis is compromised due to dysfunction of retinal pigment epithelium (RPE) cells. Whether FH contributes to AMD pathogenesis only via complement system dysregulation remains unclear. To investigate the potential role of FH on energy metabolism and oxidative stress in RPE cells, we silenced CFH in human hTERT-RPE1 cells. FH-deprived RPE cells exposed to oxidative insult, showed altered metabolic homeostasis, including reduction of glycolysis and mitochondrial respiration, paralleled by an increase in lipid peroxidation. Our data suggest that FH protects RPE cells from oxidative stress and metabolic reprogramming, highlighting a novel function for FH in AMD pathogenesis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/898551v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@32644borg.highwire.dtl.DTLVardef@569cf4org.highwire.dtl.DTLVardef@5a8aa2org.highwire.dtl.DTLVardef@43cc55_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2019

HDAC inhibition ameliorates cone survival in retinitis pigmentosa mice

Cone photoreceptor cell death in inherited retinal diseases, such as Retinitis Pigmentosa (RP), leads to the loss of high acuity and color vision and ultimately to blindness. In RP, a vast number of mutations perturb the structure and function of rod photoreceptors while cones remain initially unaffected. Cone death follows rod death secondarily due to increased oxidative stress, inflammation, and loss of structural and nutritional support provided by rods. Here, we show that secondary cone cell death in animal models for RP was associated with an increased activity of histone deacetylates (HDACs). A single intravitreal injection of an HDAC inhibitor at a late stage of the disease, when majority of rods have already degenerated, was sufficient to delay cone death and support long-term cone survival. Moreover, the surviving cones remained light sensitive and initiated light-driven ganglion cell responses. RNA-seq analysis of protected cones demonstrated that HDAC inhibition led to multi-level protection via regulation of different pro-survival pathways, including MAPK, PI3K-Akt, and autophagy. This study suggests a unique possibility for a targeted pharmacological protection of both primary degenerating rods and secondary dying cones by HDAC inhibition and creates hope to maintain vision in RP patients independent of the disease stage.

neuroscience↗