Browsing by Author "Kersey, Holly N."

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    Comparative analysis of nuclei isolation methods for brain single-nucleus RNA sequencing
    (bioRxiv, 2025-03-26) Kersey, Holly N.; Acri, Dominic J.; Dabin, Luke C.; Hartigan, Kelly; Mustaklem, Richard; Park, Jung Hyun; Kim, Jungsu; Medical and Molecular Genetics, School of Medicine
    Single-nucleus RNA sequencing (snRNA-seq) enables resolving cellular heterogeneity in complex tissues. snRNA-seq overcomes limitations of traditional single-cell RNA-seq by using nuclei instead of cells, allowing to utilize frozen tissues and difficult-to-isolate cell types. Although various nuclei isolation methods have been developed, systematic evaluations of their effects on nuclear integrity and subsequent data quality remain lacking, a critical gap with profound implications for the rigor and reproducibility. To address this, we compared three mechanistically distinct nuclei isolation strategies with brain tissues: a sucrose gradient centrifugation-based method, a spin column-based method, and a machine-assisted platform. All methods successfully captured diverse cell types but revealed considerable protocol-dependent differences in cell type proportions, transcriptional homogeneity, and the preservation of cell-type-specific and cell-state-specific markers. Moreover, isolation workflows differentially influenced contamination levels from ambient, mitochondrial, and ribosomal RNAs. Our findings establish nuclei isolation methodology as a critical experimental variable shaping snRNA-seq data quality and biological interpretation. Motivation: Single-nucleus RNA sequencing (snRNA-seq) has become an essential tool for transcriptomic analysis of complex tissues. However, the quality and efficiency of data generation depend heavily on the method used for nuclear isolation. The existing isolation techniques vary in their ability to preserve nuclear integrity, minimize ambient RNA contamination, and optimize recovery rates. Despite these differences in quality, a systematic comparison of these methods, specifically for brain tissue, is lacking. This gap poses a challenge for researchers in choosing the most suitable approach for their particular experimental requirements. To address this critical issue, our study directly compared three nuclei isolation methods and evaluated their performance in terms of yield, purity, and downstream sequencing quality. By providing a comprehensive assessment, we aim to guide researchers in selecting the most appropriate isolation protocol for their snRNA-seq experiments, ensuring optimal results and advancing the study of complex brain tissues at the single-nucleus level.
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    Control of the temporal development of Alzheimer's disease pathology by the MR1/MAIT cell axis
    (BMC, 2023-03-21) Wyatt‑Johnson, Season K.; Kersey, Holly N.; Codocedo, Juan F.; Newell, Kathy L.; Landreth, Gary E.; Lamb, Bruce T.; Oblak, Adrian L.; Brutkiewicz, Randy R.; Microbiology and Immunology, School of Medicine
    Background: Neuroinflammation is an important feature of Alzheimer's disease (AD). Understanding which aspects of the immune system are important in AD may lead to new therapeutic approaches. We study the major histocompatibility complex class I-related immune molecule, MR1, which is recognized by an innate-like T cell population called mucosal-associated invariant T (MAIT) cells. Methods: Having found that MR1 gene expression is elevated in the brain tissue of AD patients by mining the Agora database, we sought to examine the role of the MR1/MAIT cell axis in AD pathology. Brain tissue from AD patients and the 5XFAD mouse model of AD were used to analyze MR1 expression through qPCR, immunofluorescence, and flow cytometry. Furthermore, mice deficient in MR1 and MAIT cells were crossed with the 5XFAD mice to produce a model to study how the loss of this innate immune axis alters AD progression. Moreover, 5XFAD mice were also used to study brain-resident MAIT cells over time. Results: In tissue samples from AD patients and 5XFAD mice, MR1 expression was substantially elevated in the microglia surrounding plaques vs. those that are further away (human AD: P < 0.05; 5XFAD: P < 0.001). In 5XFAD mice lacking the MR1/MAIT cell axis, the development of amyloid-beta plaque pathology occurred at a significantly slower rate than in those mice with MR1 and MAIT cells. Furthermore, in brain tissue from 5XFAD mice, there was a temporal increase in MAIT cell numbers (P < 0.01) and their activation state, the latter determined by detecting an upregulation of both CD69 (P < 0.05) and the interleukin-2 receptor alpha chain (P < 0.05) via flow cytometry. Conclusions: Together, these data reveal a previously unknown role for the MR1/MAIT cell innate immune axis in AD pathology and its potential utility as a novel therapeutic target.