Single cell transcriptomics to investigate monoallelic expression as a potential trigger of Alzheimer’s disease

阿尔茨海默病(AD)是与老龄化相关的主要疾病之一，目前全球有超过3500万人受到影响，预计发病率还会增加。尽管人们对阿尔茨海默病的发病机制进行了深入的研究，但其发病机制仍不完全清楚。某些大脑区域，如颞叶，比其他区域更早受到疾病的影响，这表明某些神经元对AD病理的敏感度存在差异。这一观察结果可能是由于神经元之间基因表达的差异导致了表型的不同表达。因此，单细胞水平的研究可能揭示阿尔茨海默病发病的触发因素。随机单等位基因表达(RMAE)是一种只表达一个基因等位基因的机制。由于等位基因是随机选择的，这种基因表达模式可以在相同细胞类型的细胞之间产生变异性，并可能是使一些神经元对AD的发展比其他神经元更敏感的一种机制。RMAE已被证明与其他认知疾病有关，如精神分裂症和自闭症，以及神经发育障碍。AD相关基因在RMAE基因中显著丰富，提示AD与RMAE之间存在联系。此外，AD特征的淀粉样前体蛋白(APP)是单等位基因表达的，这可能导致不同细胞中APP的数量不同。然而，RMAE在人类神经元中的表达范围，RMAE是如何在细胞中建立的，以及RMAE调控失调的后果几乎还没有被研究过。为了研究RMAE在晚发性AD中的作用，我们首次对来自5个健康人和5个AD患者的约2000个单个神经元的全长转录本进行了测序。此外，对各自个体的外显子进行测序将使我们能够发现杂合的SNPs和RMAE。我们将建立一个计算管道，用于RMAE分析和RMAE变化的功能后果。利用大约2000个神经元的转录组数据，我们将描述这些细胞中RMAE的转录变异性和程度。我们将检验这一假设，即阿尔茨海默病患者神经元中的RMAE模式发生了变化。使用最先进的比较转录组和共表达网络分析，我们的目标是揭示RMAE变化的功能后果，这可能与AD有关。重要的是，我们将研究不同神经元类型的网络差异，以确定神经元类型的特定变化。此外，结合表观基因组、转录因子和长的非编码RNA的信息，我们还旨在为RMAE的建立以及可能导致其在AD中的失调的机制提供重要的见解。通过我们的单个神经元解析，我们希望揭开参与AD发病和进展的新的候选基因和调控机制，这些到目前为止通过对脑组织RNA进行批量测序是不可能找到的。

Alzheimer’s disease (AD) is one of the major diseases related to aging, currently affecting more than 35 million people worldwide, and expected to increase in incidences. Although being intensely investigated, the mechanisms of the onset of AD are still not fully understood. Some brain regions, such as the temporal lobe, are affected earlier by the disease than others, suggesting differences in sensitivity of certain neurons towards AD pathology. This observation might be explained by differences in gene expression between neurons leading to varying expressivity of the phenotype. Thus, investigations at the single cell level might reveal the trigger for the onset of AD. Random monoallelic expression (RMAE) is a mechanism, in which only one allele of a gene is expressed. Since the allele is randomly chosen, this gene expression mode can create variability between cells of the same cell type and might be one mechanism to render some neurons more sensitive than others to developing AD. RMAE has been shown to be involved in other cognitive diseases, such as schizophrenia and autism, and in neurodevelopmental disorders. AD-associated genes are significantly enriched among RMAE genes, suggesting a link between AD and RMAE. Moreover, the AD-characteristic amyloid precursor protein (APP) is expressed monoallelically, potentially leading to different amounts of APP in different cells. However, the extent of RMAE in human neurons, how RMAE is established in the cell, and consequences of dysregulation in RMAE have almost not been studied yet.To investigate the role of RMAE in late-onset AD, we propose to sequence for the first time the full-length transcripts of about 2000 single neurons from five healthy and five AD-affected individuals. Sequencing additionally the exomes of the respective individuals will allow us to discover heterozygous SNPs and RMAE. We will establish a computational pipeline for RMAE analysis and functional consequences of changes in RMAE. Utilizing the transcriptome data of about 2000 neurons, we will describe the transcriptional variability and the extent of RMAE in these cells. We will test the hypothesis, that patterns of RMAE are altered in neurons of individuals with AD. Using state-of-the-art comparative transcriptome and co-expression network analyses we aim to uncover functional consequences of changes in RMAE that might be related to AD. Importantly, we will investigate network differences for different neuron types to identify neuron-type specific alterations. In addition, integrating information of the epigenome, of transcription factors, and long non-coding RNAs, we also aim to provide important insights into mechanisms that establish RMAE and might lead to its dysregulation in AD. With our single neuron resolution, we expect to unravel new candidate genes and regulatory mechanisms involved in AD onset and progression, which so far were impossible to find by bulk sequencing of RNA from brain tissue.