Profiling splicing isoforms in single cells in the atherosclerosis niche

National Institute On Aging

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Abstract

We hypothesize that origin/cell-specific isoforms are produced during cellular senescence, in normal aging and in the development of atherosclerotic lesions. We also hypothesize that splicing variants have unique functions in response to inflammation and immunological changes in atherosclerosis. Relevance to the mission of the NIA Aging is a crucial risk factor for promoting atherosclerosis. Given the cell-to-cell variation and the complex interplay between cell types in atherosclerotic lesions, atherosclerosis is one of the ideal disease models for aging study. By leveraging single-cell RNA-seq and single-molecule long-read sequencing technologies, we can profile cell-specific isoform variations in normal aging and in other related disorders. The survey of new isoform variants and their functions can help understand the mechanisms of disease progression, paving the way for identifying biomarkers and therapeutic targets in age-related disorders. This project will be developed as follows: Aim 1: To elucidate isoform variations in cellular senescence in cell culture using ScNaUmi-seq. (LGG) Different from other single-cell alternative splicing protocols currently under development in our lab, the strength of ScNaUmi-seq in mapping full-length transcripts in single cells is the accurate barcode assignment to long reads. In brief, hundreds to thousands of cells are barcoded with the 10x Genomics Chromium system. After reverse transcription and cDNA preamplification, the sample is divided into two pools: one for Illumina sequencing and one for ONT Nanopore sequencing. Because sequencing error in Illumina sequencing is low, cell and transcript barcode data from Illumina sequencing is used to guide base calling in Nanopore sequencing (Fig. 1). We aim to first use ScNaUmi-seq to detect full-transcript isoforms on human fibroblasts (WI-38) at a proliferating stage and an etoposide-induced senescent stage. Through the studies in this aim, we expect to establish the ScNaUmi-seq workflow (both experimental and computational). We also expect to identify senescence-related isoforms in this framework. Aim 2: To investigate age-related isoforms in young and old mice. (LGG and TGB) A cross-sectional study during aging in male mice has shown that aging and functional decline induce redox-related metabolic remodeling in multiple organs including heart and liver (Petr et al, 2021). These adaptive changes were shown to be organ-specific and indeed are both transcriptionally and translationally regulated through multiple pathways. To investigate RNA expression and splicing variations in normal aging, we plan to perform ScNaUmi-seq in heart and liver in young (3-8 months old) and old (27-36 months old) male mice. Because non-alcoholic fatty liver disease is common in the elderly and RNA splicing regulation has been linked with disease progression (Wu et al, 2021), we will also profile isoform diversity in fatty liver. We will extend this work to high-fat dietfed mice in which fatty liver is more likely to occur. We anticipate the identification of tissue/cell-specific isoforms in heart and liver samples. By comparing young and old samples in individual tissues, we expect to acquire an age-related full-length transcriptomic profile. We expect this finding will help extend our understanding of the interplay between various pathways during aging. Aim 3: To study cell-to-cell isoform variations in atherosclerotic plaques. (LGG and LMBI) To study isoform variations in atherosclerotic plaques, we will perform ScNaUmi-seq in low-density lipoprotein receptor-deficient (Ldlr/) and apolipoprotein E-deficient (ApoE/) mice. Ldlr and ApoE knockout mice are the two most commonly used murine models for atherosclerotic studies. High-fat diet alone or combined with a single injection of recombinant adeno-associated virus 8 (rAAV8) expressing a mouse gain-of-function mutant for proprotein convertase subtilisin/kexin type 9 (PCSK9) will be used to induce atherosclerosis in these mice. We will establish a protocol for isolating viable single cells in atherosclerotic plaques in the aortic root and the aortic arch. We will then perform ScNaUmi-seq experiments on the single cell suspension to profile isoform variations in atherosclerotic plaques. We expect these experiments will shed light on our understanding of the cellular composition in atherosclerotic plaques. The identification of full-length transcripts and isoform variants will help explore the origins of foam cells, providing further insights into the molecular mechanism of plaque formation.