Human brain multi-omics to decipher major depression pathophysiology

严重抑郁障碍(MDD)是一种常见疾病，自杀风险高，社会和经济损失大， 治疗反应差。MDD患者海马神经可塑性改变，易受应激影响 对MDD患者的情绪调节和情绪记忆的建立至关重要。在细胞层面上，我们的 小组和其他人报告了MDD中颗粒神经元较少，星形胶质细胞较少，齿状回较小。那里 是关于成年人类产生新神经元的程度的相互矛盾的发现，我们展示了 这种情况发生在生命的第80个十年，而其他小组无法证实这些发现。成虫 神经发生与抑郁症表型的发病机制和抗抑郁药物的作用有关 老鼠研究。MDD的细胞和解剖改变的致病机制在很大程度上仍然存在 未知。来自三个最大的全基因组关联研究的荟萃分析确定了102个独立的 与MDD相关的变异、269个基因和15个基因组。 迫切需要使用新技术来研究人类海马区的细胞谱系。这 该项目将确定MDD和非MDD之间的差异表达蛋白(DEP)和基因(DEG) 精神控制(CTRL)海马体，在区域和单细胞水平。在匀浆的海马体组织中， 我们将使用高分辨率质谱仪(HRMS)实现我们的蛋白质组学流水线，即单核 (SN)RNA-SEQ和SnATAC(转座酶可及染色质分析)测序。在载玻片安装的组织中 我们将使用维西姆(10倍基因组)和我们定制的幻灯片序列技术，使用确定性条形码 用于空间组学测序，共定位mRNAs、ATAC和蛋白质(DBiT-seq)。 我们的先导性蛋白质组学研究表明，与CTRL相比，uMDD中DEPS较低可促进有丝分裂、分化和 防止细胞凋亡。先导SNRNA-SEQ和SnATAC-SEQ鉴定了所有类型的神经元和胶质细胞、血管系统和 未成熟的细胞团，这映射到预期的海马子区。DBiT-SEQ Pilot 研究表明，MDD与CTRL相比，RNA剪接的变异性更高，RNA速度表明转录水平较高 MDD的不稳定性导致锥体神经元团的高度患者间异质性，以及表观遗传学 ATAC分析确定了可能与MDD发病机制相关的标记。研究80MDD和80CTRL，年龄 14-90岁，男性60%，女性40%，猝死，死亡时间短，毒理清晰，尸检 间隔24小时，RNA完整性数(RIN)8，组织pH 6，我们将：1.鉴定海马区DEPS之间 MDD和CTRL应用HRMS。2.应用SnRNA--鉴定MDD和CTRL海马区的细胞团 SEQ和SnATAC-seq.3.提供第一份海马区空间转录图谱和表观基因组图谱 染色质可及性，确定区域特定的RNA动力学，并确定MDD与CTRL多聚体 应用DBiT-Seq的差异。已确定的海马细胞活性和功能的分子调节器将 为新的MDD治疗提供潜在的可药物靶点，稍后将在细胞和动物模型中进行测试。

Major depressive disorder (MDD) is a common disease with high risk of suicide, great social and economic costs, and poor treatment response. Hippocampus neuroplasticity is altered in MDD, susceptible to stress exposure and critical for mood regulation and the establishment of emotional memories in MDD. At the cellular level, our group and others reported fewer granule neurons, fewer astrocytes, and smaller dentate gyrus in MDD. There are conflicting findings about the extent to which new neurons are generated in adult humans, and we showed this happens into the eighth decade of life, while other groups were unable to confirm these findings. Adult neurogenesis is linked to both the pathogenesis of depressive phenotypes and the action of antidepressants in mouse studies. Pathogenic mechanisms underlying cellular and anatomical changes in MDD remain largely unknown. A meta-analysis from the three largest genome-wide association studies identified 102 independent variants, 269 genes, and 15 gene-sets associated with MDD. There is an urgent need for using new technologies to investigate cell lineages in human hippocampus. This project will identify differentially expressed proteins (DEPs) and genes (DEGs) between MDD and non- psychiatric control (CTRL) hippocampus, at a regional and single cell level. In homogenized hippocampus tissue, we will implement our proteomics pipeline using high resolution mass spectrometry (HRMS), single nuclei (sn)RNA-seq and snATAC (Assay for Transposase-Accessible Chromatin) sequencing. In slide-mounted tissue we will apply Visium (10X Genomics) and our custom-made slide-seq technology, using deterministic barcoding for spatial omics sequencing, co-mapping mRNAs, ATAC and proteins (DBiT-seq). Our pilot proteomics studies showed that DEPs lower in uMDD vs. CTRL promote mitosis, differentiation, and prevent apoptosis. Pilot snRNA-seq and snATAC-seq identified all types of neurons and glia, vasculature, and immature cell clusters, which mapped with Visium onto the expected hippocampus subfields. DBiT-seq pilot studies showed higher variability of RNA splicing in MDD vs. CTRL, RNA velocity revealed high transcriptomic instability in MDD leading to high inter-patient heterogeneity in the pyramidal neuron cluster, and epigenetic ATAC profiling identified markers potentially related to MDD pathogenesis. Studying 80 MDD and 80 CTRL, age 14-90 yrs., 60% males and 40% females, with sudden death, short agonal state, clear toxicology, postmortem interval<24 hrs., RNA integrity number (RIN)>8, tissue pH>6, we will: 1. Identify hippocampus DEPs between MDD and CTRL applying HRMS. 2. Identify cell clusters’ DEGs in MDD vs. CTRL hippocampus applying snRNA- seq and snATAC-seq. 3. Deliver the first hippocampus spatial transcriptomic atlas and epigenomic atlas for chromatin accessibility, identify region-specific RNA dynamics, and determine MDD vs. CTRL multiome differences applying DBiT-seq. Identified molecular regulators of hippocampus cell viability and function will provide potential druggable targets for new MDD treatments, to be later tested in cellular and animal models.