Biology subjects

Voigt, A. P.

Publications and source records attributed to Voigt, A. P..

8 recordsLinked to original sources

bioRxiv2026

Transcriptomic Characterization of Terminal Complement Complex-bound Cells in Human Choroid Using Single-Cell RNA Sequencing

Age-related macular degeneration (AMD) is among the leading causes of blindness worldwide. Early AMD is characterized by dysfunction in the choroid, including early dropout of endothelial cells and increased deposition of the complement cascade's membrane attack complex (MAC) in the choriocapillaris. In this study, we used a single-cell RNA sequencing-based approach with barcoded antibodies to measure abundance of the MAC and other surface proteins at single cell resolution on RPE/choroid samples from four aged human donor eyes. We included antibodies to detect the MAC, CD34, CD45, and complement regulators CD55 and CD59, in addition to control antibodies. Our analysis of these data revealed cell clusters with expected gene expression profiles and antibody-based detection of CD34 and CD45 congruent with transcriptome-based cell identity. We also detected surface complement regulators CD55 and CD59 across a wide variety of cell types. Across endothelial cells, surface CD55 and CD59 appeared more abundant on venous clusters, and abundance of each was correlated with expression of a third complement regulator, clusterin (CLU). The MAC was detected on a variety of cell types, but was most abundant on the surface of cells in the macrophage family, smooth muscle cells, and pericytes. We confirmed these findings by identifying MAC deposition on choriocapillaris pericytes using immunohistochemistry for MAC, endothelial, and pericyte markers. Ultimately, these data showcase a valuable new approach to analyze gene expression and surface complement in human donor eyes, and provide novel insight into patterns of MAC deposition and complement protection in the aging human choroid.

cell biology↗

bioRxiv2026

Single-Cell Gene Expression and eQTL Analyses in the Human Retina, RPE, and Choroid in Macular Degeneration

Age-related macular degeneration (AMD) is a common, complex disease affecting older individuals that can lead to severe vision loss. It is characterized by early anatomical changes in the retina, retinal pigment epithelium (RPE), and choroid, especially in the central (macular) region. AMD can progress to severe atrophy and/or pathologic angiogenesis that leads to visual decline. Over 30 genetic loci have been identified as contributing to AMD risk; however, the mechanisms by which genetic variants affect pathology has not been thoroughly explored. In this report we examined single-nucleus gene expression in the retina, RPE and choroid of 88 individuals categorized by AMD stage, as well as 36 previously published samples. Genotyping was performed on 1.8 million SNPs, with additional SNPs imputed, on each donor to identify expression quantitative trait loci (eQTLs). We found that two AMD-risk loci (PILRB and ARMS2/HTRA1) affected the expression of PILRB and HTRA1, respectively. The risk allele of PILRB was associated with increased PILRB RNA in cones, fibroblasts, choroidal resident macrophages, and RPE, whereas the HTRA1 risk locus was associated with decreased HTRA1 RNA in the RPE. We also identified an age-related decrease in complement inhibitors in the choriocapillaris, a tissue susceptible to complement mediated damage in AMD. We have made the eQTL and gene expression data fully available on the web resource Spectacle for rapid and interactive data access.

cell biology↗

bioRxiv2023

Loss of NR2E3 disrupts rod photoreceptor cell maturation causing a fate switch late in human retinal development.

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generate retinal organoids using induced pluripotent stem cells (iPSC) derived from a patient with genetic photoreceptor disease due to mutations in NR2E3, an isogenic control, and an unrelated control. Organoids were sampled using single-cell RNA sequencing across the developmental window encompassing photoreceptor specification, emergence, and maturation, up to 260 days of in vitro differentiation. Using single-cell transcriptomics data, we reconstruct the rod photoreceptor developmental lineage and identify a branchpoint in development unique to the disease state that gives rise to a divergent rod photoreceptor cell population. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including expression of the light-sensitive protein rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes at both the transcript and protein level. Using joint multimodal single-cell sequencing on late-stage retinal organoids, we further identify specific putative regulatory sites where rod-specific factors act to steer rod and cone photoreceptor cell development. Importantly, these findings are strikingly different than those observed in rodent models of disease. Together, these data provide a roadmap of human photoreceptor development and leverage patient iPSCs to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation.

developmental biology↗

bioRxiv2023

GENE EXPRESSION WITHIN A HUMAN CHOROIDAL NEOVASCULAR MEMBRANE USING SPATIAL TRANSCRIPTOMICS

Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process. In this study, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We identified genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes. Within the area of neovascularization, endothelial cells were predicted to increase expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we also investigated spatial gene expression patterns within the macular neural retina and between the macular and peripheral choroid. We recapitulated previously described regional-specific gene expression patterns across both tissues. Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

genomics↗

bioRxiv2022

Non-random distribution of mitochondrial m.3243A>G heteroplasmy in human retina and its impact on cellular phenotype

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non- random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm- derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm- derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non- random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/496449v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@10b1c41org.highwire.dtl.DTLVardef@6c2d7corg.highwire.dtl.DTLVardef@1e4d09org.highwire.dtl.DTLVardef@153a5a0_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2019

A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

The introduction of genome-wide shRNA and CRISPR libraries has facilitated cell-based screens to identify loss-of-function mutations associated with a phenotype of interest. Approaches to perform analogous gain-of-function screens are less common, although some reports have utilized arrayed viral expression libraries or the CRISPR activation system. However, a variety of technical and logistical challenges make these approaches difficult for many labs to execute. In addition, genome-wide shRNA or CRISPR libraries typically contain of hundreds of thousands of individual engineered elements, and the associated complexity creates issues with replication and reproducibility for these methods. Here we describe a simple, reproducible approach using the Sleeping Beauty transposon system to perform phenotypic cell-based genetic screens. This approach employs only three plasmids to perform unbiased, whole-genome transposon mutagenesis. We also describe a ligation-mediated PCR method that can be used in conjunction with the included software tools to map raw sequence data, identify candidate genes associated with phenotypes of interest, and predict the impact of recurrent transposon insertions on candidate gene function. Finally, we demonstrate the high reproducibility of our approach by having three individuals perform independent replicates of a mutagenesis screen to identify drivers of vemurafenib resistance in cultured melanoma cells. Collectively, our work establishes a facile, adaptable method that can be performed by labs of any size to perform robust, genome-wide screens to identify genes that influence phenotypes of interest.

genomics↗

bioRxiv2019

Src-dependent DBL family members drive resistance to vemurafenib in human melanoma

The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAFV600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/Rac1/Pak signaling axis capable of driving resistance to both current and next-generation BRAF inhibitors. However, we show that the Src inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance.

cancer biology↗

bioRxiv2018

Transcriptional heterogeneity in cancer-associated regulatory T cells is predictive of survival.

Regulatory T cells (Tregs) are a population of T cells that exert a suppressive effect on a variety of immune cells and non-immune cells. The suppressive effects of Tregs are detrimental to anti-tumor immunity. Recent investigations into cancer-associated Tregs have identified common expression patterns for tumor-infiltration, however the functional heterogeneity in tumor-infiltrating (TI) Treg is largely unknown. We performed single-cell sequencing on immune cells derived from renal clear cell carcinoma (ccRCC) patients, isolating 160 peripheral-blood (PB) Tregs and 574 TI Tregs. We identified distinct transcriptional TI Treg cell fates, with a suppressive subset expressing CD177. We demonstrate CD177+ TI-Tregs have preferential suppressive effects in vivo and ex vivo. Gene signatures derived the CD177+ Treg subset had superior ability to predict survival in ccRCC and seven other cancer types. Further investigation into the development and regulation of TI-Treg heterogeneity will be vital to the application of tumor immunotherapies that possess minimal side effects.

cancer biology↗