Implication of specific retinal cell-type involvement and gene expression changes in AMD progression using integrative analysis of single-cell and bulk RNA-seq profiling

Yafei Lyu,James A Kimble,Naifei Pan,Dwight Stambolian,Mingyao Li,Kui Wang,Christianne E Strang,Randy Zauhar,Jian Hu,Nicholas Dana,Christine A Curcio,Jeffrey D Messinger,Shanrun Liu,Paul Gamlin

doi:10.1038/s41598-021-95122-3

Abstract

Age‐related macular degeneration (AMD) is a blinding eye disease with no unifying theme for its etiology. We used single-cell RNA sequencing to analyze the transcriptomes of ~ 93,000 cells from the macula and peripheral retina from two adult human donors and bulk RNA sequencing from fifteen adult human donors with and without AMD. Analysis of our single-cell data identified 267 cell-type-specific genes. Comparison of macula and peripheral retinal regions found no cell-type differences but did identify 50 differentially expressed genes (DEGs) with about 1/3 expressed in cones. Integration of our single-cell data with bulk RNA sequencing data from normal and AMD donors showed compositional changes more pronounced in macula in rods, microglia, endothelium, Müller glia, and astrocytes in the transition from normal to advanced AMD. KEGG pathway analysis of our normal vs. advanced AMD eyes identified enrichment in complement and coagulation pathways, antigen presentation, tissue remodeling, and signaling pathways including PI3K-Akt, NOD-like, Toll-like, and Rap1. These results showcase the use of single-cell RNA sequencing to infer cell-type compositional and cell-type-specific gene expression changes in intact bulk tissue and provide a foundation for investigating molecular mechanisms of retinal disease that lead to new therapeutic targets.

Highlights

Age‐related macular degeneration (AMD) is a leading cause of legal blindness worldwide
GFAP was selectively expressed in astrocytes; PRPH, SNCG, and NEFL marked ganglion cells; NRXN2, SLC32A1, ELAVL3, and GAD1 were highly expressed in amacrine cells; VSX1 and PCP2 were selectively expressed in bipolar cells; ONECUT1, 2 and TMOD1 were selectively expressed in horizontal cells; NRL, PDE6A, GNAT1, and RHO were highly expressed in rods, and CHRNA3, TTR, GNGT2, GUCA1C, OPN1LW, and ARR3 were selectively expressed in cones
To determine if differential expression in the bulk RNA sequencing (RNA-seq) samples was due to celltype-specific differential expression and not cell-type composition, we developed a calibration-based method to detect cell-type-specific differentially expressed genes (DEGs) from bulk level gene expression for those cell-type-specific marker genes found in our scRNA-seq data (Methods)

Summary

Introduction

AMD is a leading cause of legal blindness worldwide. It affects over 10 million A mericans[1], twice the number affected by Alzheimer’s disease and equal to the total of all cancer patients combined[2], and is expected to increase as the population ages. There are 5 neuronal cell types in the retina that include photoreceptors, bipolar cells, ganglion cells, horizontal, and amacrine cells. A major glial cell, Müller, spans the retina and is involved in neurotransmission, fluid balance, and wound repair Retinal neurons and their support cells form a highly organized, vertically integrated physiologic unit. Publications of the retinal transcriptome were focused on describing the overall gene expression of the retina[11,12] and later reporting the transcriptome differences between macula and periphery using bulk RNA sequencing (RNA-seq)[13]. Cell-type-specific expression of the retina will help understand the biology of multiple diseases affecting the retina, beyond AMD

Methods

Results

Conclusion