Developing a single cell map of the aging human brain relevant to Alzheimer's disease

为了建立合适的方法学，我们最初对来自我们先前收集的背外侧前额叶皮质的16个确定的供体的180,000个核进行了单核RNA-Seq(SnRNA-Seq)。这是北美脑表达联盟的一部分，在那里我们拥有大量的基因组数据，包括全基因组测序、DNA甲基化和批量RNA-Seq，涉及250个成年人的寿命。这一数据表明，我们能够识别大多数主要的细胞类型，并能够区分具有多个已识别的抑制性和兴奋性神经元以及非神经元细胞的神经元亚型。 随后，我们从四个脑区(内嗅皮层、中回、壳核和脑室下区)获得了组织，这些组织来自一系列代表相对年轻和年长捐赠者的样本(每个区域n=6-7)。我们使用混合策略在几个实验批次中进行有效的SnRNA-Seq，然后根据已知的基因类型将池去卷积到单个供体。我们能够根据已知的标记基因区分出许多具有预期区域差异的细胞类型。年轻组和老年组之间的细胞比例似乎只有很小的差异，但随着年龄的增加，差异基因的表达出现了实质性的信号。特别是，我们注意到，虽然一些细胞类型在不同年龄组之间有基因表达下调的趋势，但另一些细胞类型的基因表达有所增加。我们特别注意到，大脑的免疫细胞显示出神经元中没有的、与年龄相关的基因表达的独特模式。 一项更大规模的后续研究已经到位，以验证包括表观遗传学测量在内的任何观察结果。

To establish appropriate methodologies, we initially performed single nuclear RNA-Seq (snRNA-Seq) on 180,000 nuclei from 16 defined donors taken from our prior collection of dorsolateral prefrontal cortex. This is part of the North American Brain Expression Consortium, where we have substantial genomic data including whole genome sequencing, DNA methylation and bulk RNA-Seq for 250 individuals across the adult human lifespan. This data demonstrated that we are able to identify most major cell types and can discriminate subtypes of neurons with multiple identified groups of both inhibitory and excitatory neurons as well as non-neuronal cells. Subsequently, we obtained tissues from four brain regions (entorhinal cortex, middle temporal gyrus, putamen and subventricular zone) from a series of samples representing relatively younger and older donors (n=6-7 per group for each region). We used a pooling strategy to perform efficient snRNA-Seq in several experimental batches then deconvoluted pools to individual donors based on known genotypes. We were able to discriminate many cell types with expected differences between regions based on known marker genes. There appear to be only modest differences in cellular proportions between the younger and older groups but substantial signal in terms of differential gene expression as a function of age. In particular, we have noted that while some cell types have a tendency to show downregulation of gene expression between age groups others show increase in expression. We particularly note that immune cells of the brain show distinct patterns of age-dependent gene expression not seen in neurons. A larger follow up study is in place to validate any observations that will also include epigenetic measurements.