Todd E. Scheetz

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.

Leishmaniasis is a group of diseases caused by the parasitic protozoa belonging to the genus, Leishmania. Visceral leishmaniasis (VL) is the severest of the syndromes, causing the majority of fatalities due to leishmaniasis. The disease is usually vector-borne, caused by a bite from a female phlebotomine sand fly. VL is prevalent in endemic regions often associated with poverty. The regions of the world where symptomatic visceral leishmaniasis occurs most commonly include India, the adjoining regions of Bangladesh and Nepal, Brazil, and countries in eastern Africa. A majority of cases are asymptomatic, leading us to question what host factors predispose individuals to develop symptomatic VL. To investigate host factors contributing to the outcome of VL, our collaborators in Varanasi, India purified CD4+ T-cells and CD14+ myeloid cells from peripheral blood or bone marrow of twelve patients with VL, prior to treatment. In parallel, control subjects from the endemic region who underwent bone marrow aspirates for unrelated conditions were also recruited. These subjects had a variety of underlying disorders and required downstream evaluation due to their final diagnoses. In this thesis we describe the evaluation of transcriptomes of the two cell types in blood and bone marrow aspirate cells from these twenty-four subjects, using RNA sequencing. These transcriptomes were aligned to the human genome assembly GRCh38 using the publicly available programs HISAT2, featureCounts, and DESeq2. The differential expression profiles validate some observations made in the literature but also raised a number of pathways and transcripts that deserve further investigation. Examples are the possible activation of the AIM2 inflammasome during infection, and the prominence of the IL17 pathway. In chapter 3 we investigated transcriptomes of both CD4+ and CD14+ cells from the same subjects, and these highlighted the possibility that some samples had been erroneously labeled at some point in the processing, necessitating removal from the final study. Pathways in the combined analyses were used to conduct pathway analyses with the Ingenuity Pathway Analysis program. Finally, the unmapped reads from the original RNA-seq alignments were investigated using the Kraken2 suite of programs and used to detect and quantify Leishmania donovani sequences in blood and bone marrow of infected subjects. Overall, the data provide insight into the biological status of subjects with VL and their infecting organisms.

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.

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors. Choroidal endothelial and immune cells are implicated in the pathogenesis of age-related macular degeneration (AMD). In a cohort of 11 AMD and 10 control human donors, Voigt et al. used single-cell RNA sequencing to identify gene expression changes across the choroidal endothelial cell and immune subpopulations. The background depicts a whole mount of human choroidal endothelial cells (red) and IBA1-expressing immune cells (green).

The m.3243A>G mutation in the mitochondrial genome commonly causes retinal degeneration in patients with maternally inherited diabetes and deafness and mitochondrial encephalopathy, lactic acidosis and stroke-like episodes. Like other mitochondrial mutations, m.3243A>G is inherited from the mother with a variable proportion of wild type and mutant mitochondrial genomes in different cells. The mechanism by which the m.3243A>G variant in each tissue relates to the manifestation of disease phenotype is not fully understood. Using a digital PCR assay, we found that the % m.3243G in skin derived dermal fibroblasts was positively correlated with that of blood from the same individual. The % m.3243G detected in fibroblast cultures remained constant over multiple passages and was negatively correlated with mtDNA copy number. Although the % m.3243G present in blood was not correlated with severity of vision loss, as quantified by Goldmann visual field, a significant negative correlation between % m.3243G and the age of onset of visual symptoms was detected. Altogether, these results indicate that precise measurement of % m.3243G in clinically accessible tissues such as skin and blood may yield information relevant to the management of retinal m.3243A>G-associated disease.