Student Theses and Dissertations

Date of Award

2024

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Cao Laboratory

Abstract

Previous single-cell methods are limited by their relatively low throughput and high costs. This prevents most laboratories from performing large-scale cell profiling, complicating the understanding of rare cell type contributions to aging and disease. Moreover, these methods often focus on the 3' end of genes. Without capturing the complete gene body coverage, these technologies have limited detection of specific isoforms. To address these limitations, we extensively optimized a combinatorial indexing-based framework to develop EasySci. We used EasySci to study cell population dynamics associated with aging and Alzheimer’s disease in the mammalian brain. This dataset recovered approximately 1.5 million single-cell transcriptomes and 400,000 chromatin accessibility profiles from 20 mouse brains. An optimized clustering framework identified more than 300 distinct cell subtypes. This extensive cataloging revealed the molecular characteristics and spatial distribution of these subtypes, providing new insights into the cellular landscape of the brain. For instance, we identified the spatial locations of astrocyte and neuron subtypes, mapping them to distinct anatomical regions. We observed cell population shifts of rare cell types during aging, i.e. an expansion in inflammatory subtypes of microglia and oligodendrocytes. Contrary, neuronal and oligodendrocyte progenitor cell populations declined. In exploring the cellular response to Alzheimer’s disease associated genetic mutations, we investigated the cell population dynamics of both established early-onset and novel late-onset Alzheimer’s disease models. This comparison highlighted the late-onset Alzheimer’s disease model, APOE*4/Trem2*R47H, having cell population dynamics reminiscent of the 5xFAD early-onset model. This suggests the novel model’s potential utility in Alzheimer’s disease research. For instance, the expansion of septal nuclei neurons were observed in both models. This neuron subtype had increased axonogenesis gene module expression. Contrary, the reduction of a choroid plexus epithelial cell subtype, marked by the enrichment of neuroprotective mitochondrial genes, were detected in both models. On a molecular level, a novel isoform of theTrem2gene was consistently upregulated in both mouse models. Furthermore, we profiled 118,240 cells from human brains. We detected region-specific transcriptomic changes in Alzheimer’s disease patients, as well as conserved trends between mouse models and human patients. This dataset also identified novel, sensitive and specific histological markers for pericytes, i.e. SLC6A12 and SLC19A1. Antibodies targeting these proteins were highly effective in staining pericytes in human brains. These experiments validated the efficacy of these markers and selected the SLC6A12 antibody as the most sensitive and specific markers of human pericytes. Furthermore these novel markers overperformed the traditionally used PDGFRB antibody. In conclusion, this dataset provided an extensive resource for understanding the cellular dynamics of aging and Alzheimer’s disease. Furthermore, we introduced EasySci, a novel, high-throughput, and cost-effective single-cell technique. This method significantly enhances the capacity of independent laboratories to profile millions of cells, and advances our understanding of normal and pathological aging.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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