Wang Yun-Mei, Wang Wen-Chao, Pan Yongzhang, Zeng Lin, Wu Jing, Wang Zheng-Bo, Zhuang Xiao-Lin, Li Ming-Li, Cooper David N, Wang Sheng, Shao Yong, Wang Li-Min, Fan Ying-Yin, He Yonghan, Hu Xin-Tian, Wu Dong-Dong
State Key Laboratory of Genetic Evolution & Animal Models, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, 650201, China.
Yunnan Key Laboratory of Biodiversity Information, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China.
Genome Med. 2025 Apr 28;17(1):41. doi: 10.1186/s13073-025-01469-x.
Deciphering the functionality and dynamics of brain networks across different regions and age groups in non-human primates (NHPs) is crucial for understanding the evolution of human cognition as well as the processes underlying brain pathogenesis. However, systemic delineation of the cellular composition and molecular connections among multiple brain regions and their alterations induced by aging in NHPs remain largely unresolved.
In this study, we performed single-nucleus RNA sequencing on 39 samples collected from 10 brain regions of two young and two aged rhesus macaques using the DNBelab C4 system. Validation of protein expression of signatures specific to particular cell types, brain regions, and aging was conducted through a series of immunofluorescence and immunohistochemistry staining experiments. Loss-of-function experiments mediated by short hairpin RNA (shRNA) targeting two age-related genes (i.e., VSNL1 and HPCAL4) were performed in U251 glioma cells to verify their aging effects. Senescence-associated beta-galactosidase (SA-β-gal) staining and quantitative PCR (qPCR) of senescence marker genes were employed to assess cellular senescence in U251 cells.
We have established a large-scale cell atlas encompassing over 330,000 cells for the rhesus macaque brain. Our analysis identified numerous gene expression signatures that were specific to particular cell types, subtypes, brain regions, and aging. These datasets greatly expand our knowledge of primate brain organization and highlight the potential involvement of specific molecular and cellular components in both the regionalization and functional integrity of the brain. Our analysis also disclosed extensive transcriptional alterations and cell-cell connections across brain regions in the aging macaques. Finally, by examining the heritability enrichment of human complex traits and diseases, we determined that neurological traits were significantly enriched in neuronal cells and multiple regions with aging-relevant gene expression signatures, while immune-related traits exhibited pronounced enrichment in microglia.
Taken together, our study presents a valuable resource for investigating the cellular and molecular architecture of the primate nervous system, thereby expanding our understanding of the mechanisms underlying brain function, aging, and disease.
解读非人灵长类动物（NHPs）不同脑区和年龄组的脑网络功能与动态，对于理解人类认知的进化以及脑发病机制至关重要。然而，NHPs多个脑区的细胞组成和分子连接的系统描绘以及衰老引起的变化在很大程度上仍未得到解决。
在本研究中，我们使用DNBelab C4系统对从两只年轻和两只老年恒河猴的10个脑区收集的39个样本进行了单核RNA测序。通过一系列免疫荧光和免疫组织化学染色实验对特定细胞类型、脑区和衰老特异性标志物的蛋白质表达进行了验证。在U251胶质瘤细胞中进行了靶向两个与年龄相关基因（即VSNL1和HPCAL4）的短发夹RNA（shRNA）介导的功能丧失实验，以验证它们的衰老效应。采用衰老相关β-半乳糖苷酶（SA-β-gal）染色和衰老标志物基因的定量PCR（qPCR）来评估U251细胞中的细胞衰老。
我们为恒河猴大脑建立了一个包含超过330,000个细胞的大规模细胞图谱。我们的分析确定了许多特定于特定细胞类型、亚型、脑区和衰老的基因表达特征。这些数据集极大地扩展了我们对灵长类动物脑组织的认识，并突出了特定分子和细胞成分在脑区域化和功能完整性中的潜在作用。我们的分析还揭示了衰老猕猴脑区广泛的转录变化和细胞间连接。最后，通过检查人类复杂性状和疾病的遗传力富集情况，我们确定神经性状在具有衰老相关基因表达特征的神经元细胞和多个区域中显著富集，而免疫相关性状在小胶质细胞中表现出明显富集。
综上所述，我们的研究为研究灵长类神经系统的细胞和分子结构提供了宝贵资源，从而扩展了我们对脑功能、衰老和疾病潜在机制的理解。
