Biology subjects

Saint-Geniez, M.

Publications and source records attributed to Saint-Geniez, M..

8 recordsLinked to original sources

bioRxiv2025

Reprogramming Factors Activate a Non-Canonical Oxidative Resilience Pathway That Can Rejuvenate RPEs and Restore Vision

Oct4, Sox2, and Klf4 (OSK) Yamanaka factors induce pluripotency and reverse age-related epigenetic changes, yet the mechanisms by which they promote rejuvenation remain poorly explored. Oxidative stress contributes to CNS aging and retinal pigmented epithelium (RPE) degeneration in age-related macular degeneration. We find that OSK expression in RPE restores retinal structure and visual function in aged mice and promotes oxidative resilience through a non-canonical, Tet2-independent pathway. Integrative functional genomics identifies GSTA4, a detoxifying enzyme that clears the lipid peroxidation byproduct 4-HNE, as a necessary and sufficient OSK effector. Dynamic GSTA4 regulation by OSK recapitulates a stem cell derived stress resilience program. GSTA4 overexpression alone enhances mitochondrial resilience, rejuvenates the aged RPE transcriptome, and reverses visual decline. GSTA4 is consistently upregulated across diverse lifespan-extending interventions suggesting a broader pro-longevity role. These findings uncover a previously unrecognized protective axis driven by Yamanaka factors that circumvents reprogramming, providing therapeutic insights for age-related diseases. HIGHLIGHTSO_LIOSK-GSTA4 provides a dynamic, Tet2-independent stress-resilience axis. C_LIO_LIFunctional genomics pinpoints GSTA4 as a direct downstream effector activated by OSK. C_LIO_LIRPE aging involves progressive accumulation of 4-HNE that can be detoxified by GSTA4. C_LIO_LIEnhancing GSTA4 rejuvenates RPE cells, restores vision and is associated with lifespan-extending interventions. C_LI

genetics↗

bioRxiv2025

Identification of cellular and molecular risk signatures for progression to late-stage age-related macular degeneration using the 9-step Minnesota Grading System

Age-related macular degeneration (AMD) is a complex multifactorial disease, and the molecular mechanisms underpinning the progression of intermediate AMD to geographic atrophy are not fully understood. To better understand mechanisms driving progression, we performed bulk RNA sequencing on dissected macular and peripheral RPE/choroid and neural retina tissue from postmortem human eyes graded using the 9-step Minnesota Grading System (MGS). Binning of intermediate AMD cases into three distinct groups (AMD3L, AMD3M, AMD3H) based on the 5-year risk of progression enabled identification of distinct gene and pathway changes associated with progression to late-stage disease. Identified changes in gene expression were validated using ELISA or histological methods. RPE-specific genes and lipid metabolic pathways showed a transient increase in AMD3L followed by a pronounced decrease in AMD3H. In AMD3H, immune response genes such as C3, TREM2, and OLR1 were upregulated when compared to AMD3L samples, as well as genes specific to Muller glia/astrocytes (NGFR, SPP1, GPX3). Our findings support complement inhibition as a promising therapeutic option for slowing conversion to advanced AMD and identify macrophage and Muller/astrocyte genes as potential cell types to target in AMD. Further, we demonstrate the value of combining emerging, outcomes-based, clinically relevant grading systems with profiling technologies to generate new insights into ocular diseases. HighlightsO_LIIntermediate AMD (AMD3) can be further divided into 3 stages - AMD3L (low risk), AMD3M (intermediate risk), AMD3H (high risk) - using the MGS9 grading system based on the risk of disease progression to late AMD. C_LIO_LIRNA sequencing of the macular RPE/choroid shows opposing changes in gene expression of multiple biological pathways for both RPE and immune cells between AMD3L and AMD3H stages. C_LIO_LIIn the macular neural retina, most biological pathways were downregulated in AMD2 (early AMD) but upregulated in AMD4 (late AMD) compared to AMD1 (non-AMD control). C_LIO_LIMolecular and cellular signatures associated with a high risk of progression to AMD4 include activation of complement C3, two subtypes of macrophages expressing either TREM2 or OLR1, and Muller glia/astrocytes as evidenced by the upregulation of GFAP and NGFR. C_LIO_LIUnderstanding the roles of these high-risk associated genes in AMD progression will facilitate the development of new treatments that prevent or delay the irreversible central vision loss in AMD patients. C_LI

neuroscience↗

bioRxiv2024

Deletion of the endothelial glycocalyx component endomucin leads to impaired glomerular structure and function

BackgroundEndomucin (EMCN), an endothelial-specific glycocalyx component, was found to be highly expressed by the endothelium of the renal glomerulus. We reported an anti-inflammatory role of EMCN and its involvement in the regulation of vascular endothelial growth factor (VEGF) activity through modulating VEGF receptor 2 (VEGFR2) endocytosis. The goal of this study is to investigate the phenotypic and functional effects of EMCN deficiency using the first global EMCN knockout mouse model. MethodsGlobal EMCN knockout mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Flow cytometry was employed to analyze infiltrating myeloid cells in the kidneys. The ultrastructure of the glomerular filtration barrier was examined by transmission electron microscopy, while urinary albumin, creatinine, and total protein levels were analyzed from freshly collected urine samples. Expression and localization of EMCN, EGFP, CD45, CD31, CD34, podocin, albumin, and -smooth muscle actin were examined by immunohistochemistry. Mice were weighed regularly, and their systemic blood pressure was measured using a non-invasive tail-cuff system. Glomerular endothelial cells and podocytes were isolated by fluorescence-activated cell sorting for RNA-seq. Transcriptional profiles were analyzed to identify differentially expressed genes in both endothelium and podocytes, followed by gene ontology analysis of up- and down-regulated genes. Protein levels of EMCN, albumin, and podocin were quantified by Western blot. ResultsEMCN-/- mice were viable with no gross anatomical defects in kidneys. The EMCN-/- mice exhibited increased infiltration of CD45+ cells, with an increased proportion of Ly6GhighLy6Chigh myeloid cells and higher VCAM-1 expression. EMCN-/- mice displayed albuminuria with increased albumin in the Bowmans space compared to the EMCN+/+ littermates. Glomeruli in EMCN-/- mice revealed fused and effaced podocyte foot processes and disorganized endothelial fenestrations. We found no significant difference in blood pressure between EMCN knockout mice and their wild-type littermates. RNA-seq of glomerular endothelial cells revealed downregulation of cell-cell adhesion and MAPK/ERK pathways, along with glycocalyx and extracellular matrix remodeling. In podocytes, we observed reduced VEGF signaling and alterations in cytoskeletal organization. Notably, there was a significant decrease in both mRNA and protein levels of podocin, a key component of the slit diaphragm. ConclusionOur study demonstrates a critical role of the endothelial marker EMCN in supporting normal glomerular filtration barrier structure and function by maintaining glomerular endothelial tight junction and homeostasis and podocyte function through endothelial-podocyte crosstalk.

pathology↗

bioRxiv2024

Use of machine learning for quantification of retinal pigment epithelium tight junctions improves assay sensitivity

The retinal pigment epithelium (RPE) is critical for maintaining outer retinal barrier homeostasis. In age-related macular degeneration (AMD), the RPE can undergo a dedifferentiation process that includes tight junction (TJ) loss and displacement of zonula occludens-1 (ZO-1), which may impair structural and functional integrity of the RPE barrier and contribute to disease pathogenesis. Our objective was to develop an automated and sensitive quantification method for TJ aberrations in an RPE immunofluorescence imaging assay, following treatment with TNF or TGF{beta}2. However, quantifying ZO-1 morphological changes in the RPE using standard image analysis methods did not provide a satisfactory assay window. To address this challenge, we developed an imaging assay to quantify ZO-1 changes using a machine learning approach, enabling enhanced phenotypic characterization of the ZO-1 changes in RPE cells and improved assay sensitivity. We were also able to capture and quantify the reversal of these changes using etanercept, an TNF inhibitor, with this imaging assay. Our findings indicated that this machine learning ZO-1 quantification assay could serve as a potential phenotypic readout for RPE dedifferentiation and enabling large-scale mechanistic studies.

bioinformatics↗

bioRxiv2024

Protective effect of P2Y receptors antagonism on stress-induced retinal degeneration

The death of retinal pigment epithelial (RPE) cells and photoreceptors (PR) is a hallmark of the progression of several degenerative ocular disorders. The precise molecular driver(s) behind RPE and PR cell death, however, remains unknown. Recent studies have suggested the involvement of ATP and purinergic signaling in the progression of age-related macular degeneration (AMD) and retinal degeneration. We have discovered that RPE cells release ATP when subjected to stress, which in turn exacerbates stress-related signaling via purinergic receptors that ultimately results in degeneration. Our findings demonstrate that blocking P2Y purinergic receptors using suramin can effectively prevent toxin-induced RPE cell death and dysfunction in vitro. Furthermore, we show efficacy of suramin in preventing photoreceptor degeneration in vivo using the RHO-P23H zebrafish model. This study reinforces the involvement of ATP and purinergic signaling in maintaining retinal health, and highlights the potential of purinergic receptor antagonism as a therapeutic strategy for retinal degeneration.

cell biology↗

bioRxiv2024

APOE Impacts Lipid Trafficking in Retinal Pigment Epithelium Cells

Age-related macular degeneration (AMD) is typified by the formation of lipid-rich drusen under the retinal pigment epithelium (RPE) layer. Apolipoprotein E (APOE) is a known genetic risk factor for AMD and a substantial component of drusen, however, the mechanism by which APOE variants contribute to AMD pathology remains unclear. APOE is the primary cholesterol and lipid transport protein of the central nervous system, as well as a component circulating lipoproteins. To better understand how APOE-dependent lipid transport may impact AMD risk, we generated isogenic APOE iPS-RPE cells expressing each of the common human APOE isoforms, as well as an APOE knockout line. APOE knockout cells showed significant morphological and barrier function deficits, suggesting that APOE is essential for RPE health. Furthermore, we observed that APOE abundance is isoform-dependent in RPE cells and that lipid transport is deficient in APOE knockout RPE cells, as well as in RPE cells expressing APOE2, a variant associated with higher risk of AMD. Contrastingly, cells expressing APOE4 seem to respond strongly to lipid challenges by upregulating APOE to support efficient lipid transport. Our results suggest that disease associated APOE variants may impact lipid transport in RPE, contributing to the formation of drusen and impairing cellular function.

cell biology↗

bioRxiv2024

Double-stranded RNA induces retinal pigment epithelium cell degeneration and inflammation

RIG-I signaling has been previously implicated as a driver of inflammation to the retinal pigment epithelium (RPE) during age-related macular degeneration (AMD). Double-stranded RNA (dsRNA) is known to initiate RIG-I signaling and lead to a type I interferon response. We show through shRNA knockdown that RIG-I is essential for initiating an interferon response in iPS-RPE in response to both synthetic dsRNA-mimetic 3p-hpRNA and the double-stranded retrotransposable element Alu. Analysis of human tissue from patients suffering from AMD show accumulation of dsRNA, peaking at the geographic atrophy (GA) stage. Using a new murine model of 3p-hpRNA subretinal challenge to RPE cells, we confirmed that accumulation of dsRNA initiates a type I interferon response, as well as RPE and photoreceptor degeneration. Although RPE response to synthetic dsRNA was acute, extensive leukocyte migration was observed. The results from this study verify the importance of RIG-I signaling in regulating inflammation in the subretinal space and implicates dsRNA accumulation as a driver of AMD pathogenesis.

cell biology↗

bioRxiv2017

Traction force screening enabled by compliant PDMS elastomers

Acto-myosin contractility is an essential element of many aspects of cellular biology, and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening (CFS).Critically, rather than the frequently employed hydrogel polyacrylamide (PAA), we fabricate these plates using polydimethylsiloxane (PDMS) rubber. Key to this approach is that the PDMS used is very compliant, with a lower-bound Youngs modulus of approximately 0.7 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells.

biophysics↗