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

为了建立适当的方法，我们最初对来自我们先前收集的背外侧前额叶皮层的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，在那里我们有大量的基因组数据，包括整个成年人寿命中250个个体的全基因组测序，DNA甲基化和批量RNA-Seq。这些数据表明，我们能够识别大多数主要的细胞类型，并可以区分神经元的亚型与多个识别组的抑制性和兴奋性神经元以及非神经元细胞。 随后，我们从代表相对年轻和老年供体的一系列样品中获得了来自四个脑区域（内嗅皮层、中颞回、壳核和室下区）的组织（每个区域每组n=6-7）。我们使用合并策略在几个实验批次中进行有效的snRNA-Seq，然后基于已知的基因型将合并物解卷积为个体供体。我们能够根据已知的标记基因区分许多具有区域间预期差异的细胞类型。年轻组和老年组之间的细胞比例似乎只有适度的差异，但在作为年龄函数的差异基因表达方面有实质性的信号。特别是，我们注意到，虽然一些细胞类型具有在年龄组之间显示基因表达下调的趋势，但其他细胞类型显示表达增加。我们特别注意到，大脑的免疫细胞显示出在神经元中没有看到的年龄依赖性基因表达的独特模式。 一项更大的后续研究正在进行中，以验证任何观察结果，其中还包括表观遗传测量。

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.