Map Brain Disorders with Tissue-Specific Molecular Insights

The onset and progression of neurological and psychiatric disorders are intricately tied to the molecular processes within brain tissues. Research has demonstrated that the levels of transcription and protein, irrespective of the presence of post-translational modifications (PTMs), exhibit a high degree of specificity in brain tissues, making it challenging to use peripheral tissues as a proxy [1, 2]. Non-primates and non-human primates have been used to study the physiology and pathophysiology of the human brain. While the findings have been informative, significant gaps remain in accurately simulating the human brain, particularly for high-level cognitive functions such as self-cognition and theory of mind [3,4,5]. Furthermore, model animals often exhibit limited interspecies conservation in intergenic regions [3, 6], restricting the understanding of noncoding regulations. Therefore, a human-based molecular atlas that is specific to brain tissues or cell types is indispensable [2].

Population-level molecular atlases for humans, such as the 1000 Genomes, have greatly propelled biomedical research by providing a deep catalog of genetic diversity across global populations [7,8,9]. However, the biology of regulation is missing when solely establishing genetic variant-disease associations, without considering regulatory molecules. A population-level multi-omics reference panel would be fundamental for elucidating the distribution and diversity of molecules, their interrelationships, and their associations with aging and various diseases [1, 10,11,12]. A multi-omics reference panel for human brain tissues was lacking until recent efforts through projects, such as GTEx, ROSMAP, PsychENCODE, MSBB, and the study by the Knight-ADRC [1, 3, 13,14,15]. The GTEx project has constructed a joint genome-transcriptome atlas, showing transcriptional distribution across 50 human tissues and revealing the effects of genetic variation on transcriptional regulation [1]. This has facilitated the discovery of potential regulatory molecules that mediate the effects of variants on diseases, as identified from genome-wide association studies (GWAS), thus partially addressing the “missing biology” between variants and diseases [1]. However, the sample sizes for brain tissues in GTEx are limited, with only 100–200 sample, most of which are predominantly from individuals of European ancestry [1]. The Religious Orders Study/Memory and Aging Project (ROSMAP) has analyzed the genomes, epigenomes, transcriptomes, and proteomes of hundreds of human brain tissue samples, primarily from individuals of European ancestry, thereby creating a multi-omics atlas mainly focused on understanding the etiology of Alzheimer’s disease (AD) [10, 11, 13, 16,17,18]. The PsychENCODE project aims to uncover the genetic and molecular mechanisms of psychiatric disorders through omics data from brain tissues, yet it has a limited representation of Asian samples [19,20,21,22]. The Mount Sinai/JJ Peters VA Medical Center Brain Bank (MSBB–Mount Sinai NIH Neurobiobank) integrates multiple omics data across various brain regions alongside quantitative measures of neuritic plaque density and clinical dementia ratings to advance understanding of Alzheimer’s disease pathogenesis [15]. Our companion study has suggested that the molecular mechanisms underlying neuropsychiatric disorders might differ between Asian and European populations [23]. It also underscored that increasing genetic ancestral diversity is more efficient for power improvement for probing trait-associated genetic elements than increasing the sample size within single-ancestry reference panel [23]. Therefore, there is an urgent need to fill the gap in large-scale multi-omics brain atlases for Asian populations.

Due to the unique nature of brain tissues, obtaining population-scale samples through hospital- or community-based recruitment is very challenging. National brain banks offer a great opportunity for enabling systematic profiling of the human brain molecular features [24]. Led by the National Health and Disease Human Brain Tissue Resource Center and in collaboration with multiple centers within the China Human Brain Bank Consortium, we propose the China Brain Multi-omics Atlas Project (CBMAP). In Phase I of the project, we collected brain tissue samples from over 1000 Chinese donors. This project will provide a comprehensive landscape for the molecular profiles across the genome, epigenome, transcriptome, proteome, and metabolome for human brain samples (Fig. 1). It is worth mentioning that CBMAP will also encompass multiple PTMs that have not been captured in any of the previous studies at the population scale. PTMs are critical mechanisms of protein functional regulation, altering protein properties, localization, stability, and interactions through the addition or removal of specific chemical groups, or through protein cleavage [25, 26]. These modifications play essential roles in maintaining dynamic cellular functions and biological processes. Changes in the phosphorylation of tau protein are among the most characteristic manifestations of AD [27]. In addition, we will generate spatial omics and single-nucleus 3D structure of chromatin data for selected samples to provide a higher resolution and deeper insights into the molecular underpinnings of brain-related disorders. The 3D structure of chromatin is critical for gene regulation and cellular function. Chromatin regulates precise gene expression through topologically associating domains (TADs) and spatial interactions between enhancers and promoters [28]. These mechanisms are especially important in the complex development and function of the brain. Given the currently limited research on brain PTMs and 3D chromatin structures, unknown mechanisms could be revealed for the etiology and progression of brain diseases by a comprehensive study.

CBMAP is building a multi-omics reference map of over 1000 human brains (Phase I), aimed at understanding the molecular networks and features of cognition, aging, and brain-related disorders based on the population level profile, supporting the China Brain Project (CBP) [5].

Sample and tissue collection

As illustrated in Fig. 2, Phase I of the CBMAP was initiated by the China Human Brain Bank Consortium following a standardized operational protocol for human brain banking [29,30,31]. Members of the consortium including the National Health and Disease Human Brain Tissue Resource Center at Zhejiang University (ZJU) in southeastern China, the National Human Brain Bank for Development and Function at Peking Union Medical College (PUMC) in northern China, and the Xiangya Medical School Brain Bank at Central South University (CSU) in central China take part in the Phase I of the CBMAP, contributing over 1000 donors in total. These three brain banks are among the earliest established and currently hold the top-ranked brain sample collections in China. The Phase I collection covers the Yangtze River Delta urban cluster by ZJU in the southeast, the Beijing-Tianjin-Hebei metropolitan area by PUMC in the north, and the Central Yangtze River region by CSU in the middle, encompassing populations of 111 million, 245 million, and 110 million, respectively, for a total population coverage of approximately 466 million people (2022 China Urban Development Potential Ranking).

All donors or their families provided informed consent through voluntary donation agreements, permitting the use of their information and biospecimens for future studies. All procedures involving human participants were performed in accordance with the ethical guidelines of the institutional and national research regulations. The study protocol was approved by the Ethics Committee of Zhejiang University School of Medicine (2020–005 and 2024–007). During sample collection, we recorded the donor’s age, sex, time of death, and disease history. This project only included samples with an ischemia time of less than 24 h and excluded samples with highly pathogenic infections such as HIV. Donors under 18 years of age were not included in this project. Fetal and child donors will be incorporated in a future extension of the CBMAP, focusing on developmental processes.

In Phase II, the sample size will be expanded to approximately 2000 donors, by incorporating contributions from members of the China Human Brain Bank Consortium, including the Hebei Medical University Brain Bank in northern China, the Fudan University Brain Bank, and the Anhui Medical University Brain Bank in eastern China [32]. We are also actively working to integrate brain banks from the southwestern and northwestern regions of China as well as remote areas, e.g. Hainan, into the project to ensure comprehensive representation of most, if not all, populations in China.

In Phase I of the CBMAP, we prioritized the region at the intersection of Brodmann Area 9 (BA9) and the superior frontal gyrus. This region primarily overlaps with the dorsolateral prefrontal cortex (DLPFC), which is instrumental in managing various cognitive processes, including working memory, cognitive flexibility, and planning [33]. The broad functional spectrum of DLPFC implicates it in neurodegenerative diseases such as Alzheimer’s disease and a range of psychiatric disorders [3]. The DLPFC has been a focal point for several large-scale projects, including ROSMAP, PsychENOCDE, and GTEx, have also considered this brain region as a primary focus of their research [1, 3, 17]. In subsequent phases of the CBMAP, we will extend our research to encompass additional brain regions including hippocampus, striatum, amygdala, substantia nigra, and hypothalamus, thereby constructing a more comprehensive molecular atlas.

Neuropathological diagnosis

Samples collected in this project come with definitive pathological diagnoses, including primary age-related tauopathy (PART), limbic-predominant age-related TDP-43 encephalopathy (LATE), aging-related tau astrogliopathy (ARTAG), Alzheimer’s disease neuropathological change (ADNC), Lewy body disease (LBD), cerebrovascular diseases (CVD), and pathologically healthy controls. These uniformly applied pathological diagnostic procedures are organized by the National Health and Disease Human Brain Tissue Resource Center at Zhejiang University, following a unified process of quality controls [32, 34,35,36]. The neuropathological diagnoses were conducted by examining multiple brain regions based on standard procedures [34], and are not limited to the prefrontal cortex. The molecular features of pathological conditions and the pathological condition-specific regulation patterns (including genetic regulations) will be studied.

Genomics and epigenomics

We will conduct whole-genome sequencing (WGS). Considering factors such as sample size, frequency of rare variants, and sequencing costs, the sequencing depth for Phase I is set at between 10–20×. DNA methylation will be assessed using the Infinium MethylationEPIC v2.0 BeadChip. Additionally, we will carry out single-nucleus ATAC sequencing (snATAC-seq) on selected samples to capture cell-type-specific chromatin accessibility. To explore interactions among regulatory elements, we plan to conduct capture-Hi-C [37]. Moreover, we will employ a single-cell/nucleus tri-omic approach ChAIR (chromatin accessibility, interaction, and RNA simultaneously) for single-cell 3D epigenomic and transcriptional regulatory panoramic scanning [38].

To profile the transcriptome, we will conduct ribo-free bulk RNA-seq to cover as many samples as possible with RIN ≥ 5. In addition, we will conduct single-nucleus RNA sequencing (snRNA-seq) for selected samples to profile cell-type-specific regulations and to support the deconvolution-based cell-type proportion and cell-type level gene expression estimation. Random primers-based techniques including snRandom-seq will be implemented to capture the low-quality RNAs [39]. Long-read RNA-seq will be utilized to achieve a higher quality of identification of splicing events [40, 41]. Furthermore, we are conducting spatial transcriptomics on representative samples and regions to study the etiology of neurodegenerative diseases [42].

The representative samples are selected based on research questions. To probe disease-related transcriptomic features, we will select cases with clear pathological/clinical diagnoses (including different disease stages and comorbidities) and control samples matched by potential confounding factors. For identifying the molecular features of natural physiological processes (e.g., aging), we will select samples based on the distribution of age, sex, and region. These two parts will cover 100–200 samples of prefrontal cortex.

Proteomics, post-translational modifications, and metabolomics

Protein and post-translational modification (PTM) abundance will be measured using Liquid Chromatography-Mass Spectrometry (LC-MS). This will enable us to investigate protein and PTM profiles of serine/threonine phosphorylation, ubiquitination, and acetylation modifications. Additionally, we plan to examine other PTMs including lactylation to understand the hypoxia-related molecular consequences. For the samples planned for spatial transcriptomics and single-nucleus transcriptomics, we will also conduct spatial proteomics and PTM (at least for phosphorylomics) measurements to study the potential molecular functions within specific microenvironments across multiple omics layers.

In addition, we plan to perform metabolomics analysis on brain tissue homogenates and high-resolution spatial metabolomics. Homogenate metabolomics will leverage widely targeted LC-MS technology, complemented by targeted metabolomics for verification.

We will develop a data visualization portal to facilitate access by the scientific community. Results generated from the analysis, such as QTL signals and case-control differential expression, will be made available on the portal, with interactive query and download functionalities.

In Phase I, we collected tissue samples from 1187 Chinese donors from the brain banks at Zhejiang University (N = 443), Peking Union Medical College (N = 568), and Central South University (N = 176). In the pilot stage of Phase I, we have completed the bulk-level measurement for the genome, epigenome, transcriptome, proteome, and phosphoproteome profile in a subset of samples (Fig. S1) and established the data processing and quality control pipeline. High-throughput data generation is underway for all available samples. In addition, we are generating snRNA-seq data, snATAC-seq data, and single nucleus-level 3D epigenomic and transcriptional regulatory maps for representative tissue samples, with particular interest in neurodegenerative diseases including AD. Moreover, we have finished pathological diagnoses including PART, LATE, ARTAG, ADNC, LBD, and CVD for most samples.