Biology subjects

Twarog, M.

Publications and source records attributed to Twarog, M..

4 recordsLinked to original sources

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

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

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↗