Single-Mitochondrion Sequencing Uncovers Distinct Mutational Patterns and Heteroplasmy Landscape in Mouse Astrocytes and Neurons

BACKGROUND: Mitochondrial (mt) heteroplasmy can cause adverse biological consequences when deleterious mtDNA mutations accumulate disrupting ‘normal’ mt-driven processes and cellular functions. To investigate the heteroplasmy of such mtDNA changes we developed a moderate throughput mt isolation procedure to quantify the mt single-nucleotide variant (SNV) landscape in individual mouse neurons and astrocytes In this study we amplified mt-genomes from 1,645 single mitochondria (mts) isolated from mouse single astrocytes and neurons to 1. determine the distribution and proportion of mt-SNVs as well as mutation pattern in specific target regions across the mt-genome,2. assess differences in mtDNA SNVs between neurons and astrocytes, and 3. Study cosegregation of variants in the mouse mtDNA. RESULTS: 1. The data show that specific sites of the mt-genome are permissive to SNV presentation while others appear to be under stringent purifying selection. Nested hierarchical analysis at the levels of mitochondrion, cell, and mouse reveals distinct patterns of inter- and intra-cellular variation for mt-SNVs at different sites. 2. Further, differences in the SNV incidence were observed between mouse neurons and astrocytes for two mt-SNV 9027:G>A and 9419:C>T showing variation in the mutational propensity between these cell types. Purifying selection was observed in neurons as shown by the Ka/Ks statistic, suggesting that neurons are under stronger evolutionary constraint as compared to astrocytes. 3. Intriguingly, these data show strong linkage between the SNV sites at nucleotide positions 9027 and 9461. CONCLUSION: This study suggests that segregation as well as clonal expansion of mt-SNVs is specific to individual genomic loci, which is important foundational data in understanding of heteroplasmy and disease thresholds for mutation of pathogenic variants. From 13 mice, 59 neuron cells and 43 astrocytes were harvested by micropipetting. On avarage, from a single neuron or astrocyte 16 mitochondria were sampled, and in total 1645 single mitochondria were collected. In parallel, 73 control samples were made on various conditions: (1) no PCR primers, (2) no input mtDNA, (3) both (1) and (2), (4) with PCR primers and with input mtDNA but pooling multiple mitochondria. Condition (1-3) serves as negative control while (4) serves as positive control. In total, there are 179 libraries and 1717 samples including controls.

背景：线粒体异质性（mitochondrial heteroplasmy）当有害线粒体DNA（mitochondrial DNA, mtDNA）突变累积并破坏“正常”线粒体驱动的生理过程与细胞功能时，可引发不良生物学后果。为探究此类mtDNA变化的异质性，我们开发了一种中等通量线粒体分离流程，以定量分析单个小鼠神经元与星形胶质细胞中的线粒体单核苷酸变异（single-nucleotide variant, SNV）谱。本研究中，我们对从小鼠单个星形胶质细胞和神经元中分离出的1645个单个线粒体的线粒体基因组进行扩增，以达成三个研究目标：1. 明确线粒体单核苷酸变异（mitochondrial single-nucleotide variant, mt-SNV）在线粒体基因组特定靶区域的分布、占比及突变模式；2. 评估神经元与星形胶质细胞之间mtDNA单核苷酸变异的差异；3. 研究小鼠mtDNA中变异的共分离现象。
结果：1. 数据显示，线粒体基因组的特定位点允许单核苷酸变异出现，而其他位点则似乎受到严格的纯化选择。在线粒体、细胞、小鼠三个层面开展的嵌套层级分析揭示了不同位点的mt-SNV在细胞间与细胞内的变异模式存在显著差异。2. 进一步观察发现，小鼠神经元与星形胶质细胞之间的SNV发生率存在差异，涉及两个mt-SNV：9027:G>A和9419:C>T，这表明这两种细胞类型的突变倾向有所不同。通过Ka/Ks统计值可观测到神经元中存在纯化选择，这提示相较于星形胶质细胞，神经元受到更强的进化约束。3. 值得注意的是，本研究数据显示核苷酸位点9027与9461处的SNV位点之间存在强连锁关系。
结论：本研究表明，mt-SNV的分离与克隆扩增具有基因组位点特异性，这为理解致病性突变的异质性与疾病阈值提供了重要的基础数据。本研究通过显微吸移法从13只小鼠体内采集了59个神经元细胞与43个星形胶质细胞。平均而言，从单个神经元或星形胶质细胞中可采集到16个线粒体，总计收集到1645个单个线粒体。同时，我们设置了73份不同条件的对照样本：(1) 未添加PCR引物；(2) 未加入输入型mtDNA；(3) 同时满足(1)和(2)的条件；(4) 添加PCR引物与输入型mtDNA，但混合多个线粒体。其中条件(1)-(3)作为阴性对照，条件(4)作为阳性对照。最终总计获得179个测序文库与1717个样本（含对照样本）。