Biomarker Identification for Gender Specificity of Alzheimer's Disease Based on the Glial Transcriptome Profiles

Published: May 20, 2024

doi:

Hanjie Liu*¹, Hui Yang*², Shuqing Liu, Siyu Li, Wen Yang, Anwar Ayesha, Xin Tan, Cen Jiang, Yi Liu, Lushuang Xie

¹Chengdu University of Traditional Chinese Medicine, ²Chengdu Shuangliu Hospital of Traditional Chinese Medicine, ³Department of Automation,Tsinghua University, ⁴Affiliated Reproductive & Women-Children Hospital,Chengdu University of Traditional Chinese Medicine, ⁵Department of Neurology, Dalian Municipal Central Hospital,Central Hospital of Dalian University of Technology

Summary

This study analyzed single-nuclei transcriptomes of thirty-three individuals with Alzheimer’s disease (AD), revealing sex-specific DEGs in glial cells. Functional enrichment analysis highlighted synaptic, neural, and hormone-related pathways. Key genes, namely NLGN4Y and its regulators, were identified, and potential therapeutic candidates for gender-specific AD were proposed.

Abstract

Many sex-specific biomarkers have been recently revealed in Alzheimer's disease (AD); however, cerebral glial cells were rarely reported. This study analyzed 220,095 single-nuclei transcriptomes from the frontal cortex of thirty-three AD individuals in the GEO database. Sex-specific Differentially Expressed Genes (DEGs) were identified in glial cells, including 243 in astrocytes, 1,154 in microglia, and 572 in oligodendrocytes. Gene Ontology (GO) functional annotation analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed functional concentration in synaptic, neural, and hormone-related pathways. Protein-protein interaction network (PPI) identified MT3, CALM2, DLG2, KCND2, PAKACB, CAMK2D, and NLGN4Y in astrocytes, TREM2, FOS, APOE, APP, and NLGN4Y in microglia, and GRIN2A, ITPR2, GNAS, and NLGN4Y in oligodendrocytes as key genes. NLGN4Y was the only gene shared by the three glia and was identified as the biomarker for the gender specificity of AD. Gene-transcription factor (TF)-miRNA coregulatory network identified key regulators for NLGN4Y and its target TCMs. Ecklonia kurome Okam (Kunbu) and Herba Ephedrae (Mahuang) were identified, and the effects of the active ingredients on AD were displayed. Finally, enrichment analysis of Kunbu and Mahuang suggested that they might act as therapeutic candidates for gender specificity of AD.

Introduction

Alzheimer's disease (AD) is a global disease with high incidence, and it accounts for 60%-80% of dementia¹. Despite its high incidence, the mechanistic pathogenesis of AD is not clearly delineated, and there have been no effective therapeutics until now². The main pathologies in AD were identified as neuronal atrophy and the accumulation of pathological debris, mainly microtubule-associated protein Tau, and β-amyloid (Aβ)³^,⁴. The pathogenesis of AD is associated with abnormal autophagy, oxidative stress, mitochondrial dysfunction, inflammation, and energy metabolism disorder⁵. Prevalence surveys proved that two-thirds of AD patients were women⁶. Sex-specific differences in AD exist in the etiology, clinical manifestations, prevention, and treatment. Thus, revealing the biological mechanism that causes sex-specific differences in AD and targeting traditional Chinese medicine (TCM) can potentially provide a more comprehensive theoretical framework to understand the pathogenesis of AD, and to further guide accurate treatment strategy.

Neuroglial cells, especially microglia, astrocytes, and oligodendrocytes, potentially contribute to the pathogenesis of AD. In AD, microglia are activated and genetically altered, which contribute to inflammatory response, phagocytosis, and Aβ clearance⁷^,⁸; astrocyte is genetically altered, which affects synaptic activity, ion homeostasis, and energy and lipid metabolism⁹; oligodendrocyte is genetically altered with sex specificity, which contributes to neuronal loss, neurofibrillary tangles, and white matter lesions¹⁰^,¹¹.

In this study, we employed single-nuclei RNA sequencing (snRNA-seq) as a superior technique. Compared to single-cell RNA sequencing (scRNA-seq), snRNA-seq offers advantages in terms of sample richness, cell type integrity, and data reliability¹²^,¹³. SnRNA-seq has been extensively utilized in studies focusing on AD and exploring the role of glial cells¹⁴^,¹⁵^,¹⁶. Its wide adoption in these research areas highlights its effectiveness in providing valuable insights into the transcriptional characteristics of glial cells in AD. By leveraging the advantages of snRNA-seq, researchers have been able to uncover crucial information regarding the involvement of glial cells in AD pathology and identify potential therapeutic targets. In order to explore sex-specific neuroglial transcriptional characteristics in AD and potential TCMs for sex specificity of AD, this study analyzed snRNA-seq data from the frontal cortex of AD patients from the NCBI GEO public database. Sex-specific differentially expressed genes (DEGs), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Protein-protein interaction (PPI) network, and gene-TF-miRNA network are further analyzed to reveal key biomarkers and potential pathogenesis. Finally, potential TCMs were suggested, and their active ingredients were displayed with tables by searching the Coremine Medical, TCMIP, and TCMSP databases.

Protocol

Steps 2 to 9 of the analysis were implemented using R software (see Supplementary Figure 1 and Supplementary File 1), while the remaining steps were executed on the online platforms. The details of the databases used in this protocol (along with the weblinks) are provided in the Table of Materials. 1. Data acquisition Access the publicly available Gene Expression Omnibus (GEO) database at the Natio…

Representative Results

SnRNA-seq analysis of frontal glial transcriptome profiles and the annotation of cell types In total, 220,095 nuclei and 32,077 genes in the frontal cortex of 17 male AD and 17 female AD were obtained (Figure 1A). UMAP plot visualized the total single-nuclei frontal transcriptomes displaying distinct types of nuclei after dimension reduction analysis (Figure 1B). Total numbers of annotated nuclei captured by gender were shown, which m…

Discussion

Gender-specificity has been identified in epidemiology, pathology, and clinical manifestation of AD¹⁹. Here, we confirmed the potential pathological mechanism of the “hormone-synapse-neuron axis” from gender-specific glial genes and related pathways in AD patients. NLGN4Y was the only shared gene in the three glia and was chosen as the biomarker for the gender specificity of AD. TF and miRNAs regulating NLGN4Y were strongly linked to gender differences and the development of the nervous system. Fu…

Declarações

The authors have nothing to disclose.

Acknowledgements

The authors are grateful to Jessica S Sadick, Michael R O'Dea, Philip Hasel, etc., for providing the GSE167490 dataset. The authors appreciate that Faten A Sayed, Lay Kodama, Li Fan, etc., offer the GSE183068 dataset. The authors thank Shuqing Liu for the help with data analysis and Wen Yang for providing the data analysis platform. This study was supported by National Natural Science Foundation of China (82174511), Chengdu University of Traditional Chinese Medicine Apricot Grove Scholars, Discipline Talent Research Enhancement Program (QJJJ2022001), LiaoNing Revitalization Talents Program (XLYC 1807083), Sichuan Administration Bureau Fund of Chinese Medicine and Herbs (2023MS578), National Undergraduate Innovation and Entrepreneurship Training Project (202310633003X), and Innovative topics of scientific research practice for college students in Chengdu University of Traditional Chinese Medicine (ky-2023100). Hanjie Liu and Hui Yang contributed to the design of the study, collection, interpretation of data, and drafting, and revising the manuscript. Shuqing Liu and Siyu Li participated in the design of the study, collection of data and drafting the manuscript. Wen Yang and Anwar Ayesha were responsible for the collection and interpretation of data. Xin Tan prepared figures and/or tables. Cen Jiang, Yi Liu, and Lushuang Xie conceived the study and reviewed/edited the manuscript. All authors contributed to the article and approved the submitted version.

Materials

Database
Coremine Medical database	Jointly developed by Norway, the Chinese Academy of Sciences, the Chinese Academy of Medical Sciences, the National Medical Library of the United States and other institutions	When you explore concepts in CoreMine Medical you access a database that is structured to relate important concepts, ranked by statistical relevance, to your topic. For example, if you type in "Alzheimer disease," in addition to retrieving documents and resources that discuss the disease, you will be able to view networks and lists that show how your query concept is related to other bio-medical concepts. This provides an overview of concepts that relate to your search as well as being an interface for navigating information on these concepts. Weblink: https://coremine.com/medical/
Gene Expression Omnibus (GEO)	National Center for Biotechnology Information in the United States (NCBI)	GEO is a public functional genomics data repository supporting MIAME-compliant data submissions. Array- and sequence-based data are accepted. Tools are provided to help users query and download experiments and curated gene expression profiles. Weblink: https://www.ncbi.nlm.nih.gov/geo/
Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine (TCMIP, version: 2.0)	None	Introduction to the Integrated Pharmacology Based Network Computational Research Platform for Traditional Chinese Medicine [TCMIP v2.0], http://www.tcmip.cn/ ) It is an intelligent data mining platform based on the online database of the Encyclopedia of Traditional Chinese Medicine (ETCM), which integrates medical big data management and pharmacological computing services. It aims to reveal the scientific connotation of traditional Chinese medicine theory and the scientific value of original thinking in traditional Chinese medicine, summarize and pass on the experience of famous doctors, control the quality of traditional Chinese medicine, explain the principles of traditional Chinese medicine action, research and development of new Chinese medicine, especially the discovery and optimization of modern drug combinations, Provide a strong data foundation and analytical tools. Based on TCMIP v1.0, a comprehensive upgrade is implemented, including five major databases and seven functional modules. Through system integration and module integration, a comprehensive analysis of the multi-level correlation of the "disease syndrome prescription" interaction network can be quickly achieved. As an intelligent data mining platform, TCMIP v2.0 will provide a strong data foundation and analysis platform for revealing the scientific connotation of traditional Chinese medicine theory and the scientific value of original thinking in traditional Chinese medicine, summarizing and inheriting the experience of famous doctors, quality control of traditional Chinese medicine, elucidating the principles of traditional Chinese medicine action, research and development of new traditional Chinese medicine drugs, especially modern drug combination discovery and optimization. Weblink: http://www.tcmip.cn/TCMIP
NetworkAnalyst	None	Networkanalyze is an online visualization analysis platform for gene expression analysis and meta-analysis. It can perform comparative, quantitative, differential and enrichment analysis of gene expression, protein-protein interaction analysis, integration analysis of multiple datasets, and can also draw high-value images such as PCA, protein-protein interaction network diagram, heatmap, volcano diagram, Wayne diagram, etc. Weblink: https://www.networkanalyst.ca/NetworkAnalyst/
PubMed database	National Center for Biotechnology Information in the United States (NCBI)	The Pubmed database is a biomedical literature database maintained by the National Library of Medicine (NLM) in the United States, aimed at providing the latest medical research results to scientists, doctors, researchers, and students worldwide. This database collects biomedical literature from around the world, including journal articles, papers, books, etc. As of now, the Pubmed database has collected over 30 million articles and is continuously updated every week. Weblink: https://pubmed.ncbi.nlm.nih.gov/
R software	Ross Ihaka and Robert Gentleman	R is a language and environment for statistical computing and graphics. It is a GNU project which is similar to the S language and environment which was developed at Bell Laboratories (formerly AT&T, now Lucent Technologies) by John Chambers and colleagues. R can be considered as a different implementation of S. There are some important differences, but much code written for S runs unaltered under R. Weblink: https://www.r-project.org/
STRING database (STRING, version 11.0)	Swiss Institute of Bioinformatics	STRING is a database of known and predicted protein interactions. The interactions include direct (physical) and indirect (functional) associations Weblink: https://string-db.org/
Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP)	Zhejiang Jiuwei Health Co., Ltd	TCMSP is not only a data repository, but also an analysis platform for users to comprehensively study Traditional Chinese Medicines (TCM): including identification of active components, screening of drug targets and generation of compounds-targets-diseases networks, as well as the detailed drug pharmacokinetic information involving drug-likeness (DL), oral bioavailability (OB), blood-brain barrier (BBB),intestinal epithelial permeability (Caco-2), ALogP,fractional negative surface area (FASA-) and number of H-bond donor/acceptor (Hdon/Hacc). So far, TCMSP has attracted broad attentions and several groups have published more than 10 papers by using our TCMSP database within about one year. Weblink: https://tcmsp-e.com

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