The accumulation of mtDNA mutations in different tissues from various mouse models has been widely studied especially in the context of mtDNA mutation-driven ageing but has been confounded by the inherent limitations of the most widely used approaches. By implementing a method to sequence mtDNA without PCR amplification prior to library preparation, we map the full unbiased mtDNA mutation spectrum across six distinct brain regions from mice. We demonstrate that ageing-induced levels of mtDNA mutations (single nucleotide variants and deletions) reach stable levels at 50 weeks of age but can be further elevated specifically in the cortex, nucleus accumbens (NAc), and paraventricular thalamic nucleus (PVT) by expression of a proof-reading-deficient mitochondrial DNA polymerase, PolgD181A. The increase in single nucleotide variants increases the fraction of shared SNVs as well as their frequency, while characteristics of deletions remain largely unaffected. In addition, PolgD181A also induces an ageing-dependent accumulation of non-coding control-region multimers in NAc and PVT, a feature that appears almost non-existent in wild-type mice. Our data provide a novel view of the spatio-temporal accumulation of mtDNA mutations using very limited tissue input. The differential response of brain regions to a state of replication instability provides insight into a possible heterogenic mitochondrial landscape across the brain that may be involved in the ageing phenotype and mitochondria-associated disorders.

中文翻译：

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mtDNA突变谱的无偏差无PCR时空图揭示了大脑区域对复制不稳定性的特异性反应

广泛研究了来自各种小鼠模型的不同组织中mtDNA突变的积累，尤其是在mtDNA突变驱动的衰老背景下，但是由于最广泛使用的方法的固有局限性而感到困惑。通过实施在不进行PCR扩增之前就对mtDNA进行测序的方法，我们可以在小鼠的六个截然不同的大脑区域上绘制完整的mtDNA突变谱图。我们证明了衰老诱导的mtDNA突变水平（单核苷酸变异和缺失）在50周龄时达到稳定水平，但可以通过表达进一步在皮质，伏隔核（NAc）和脑室丘脑旁核（PVT）中进一步升高校正缺陷的线粒体DNA聚合酶PolgD181A。单核苷酸变体的增加增加了共享的SNV的比例及其频率，而缺失的特征在很大程度上未受影响。此外，PolgD181A还诱导了NAc和PVT中非编码控制区多聚体的衰老依赖性积累，这一特征在野生型小鼠中几乎不存在。我们的数据提供了使用非常有限的组织输入的mtDNA突变的时空累积的新颖观点。大脑区域对复制不稳定状态的差异响应提供了对大脑中可能与年龄表型和线粒体相关疾病有关的异源线粒体景观的见解。PolgD181A还诱导NAc和PVT中非编码控制区多聚体的衰老依赖性积累，这一特征在野生型小鼠中几乎不存在。我们的数据提供了使用非常有限的组织输入的mtDNA突变的时空累积的新颖观点。大脑区域对复制不稳定状态的差异响应提供了对大脑中可能与年龄表型和线粒体相关疾病有关的异源线粒体景观的见解。PolgD181A还诱导NAc和PVT中非编码控制区多聚体的衰老依赖性积累，这一特征在野生型小鼠中几乎不存在。我们的数据提供了使用非常有限的组织输入的mtDNA突变的时空累积的新颖观点。大脑区域对复制不稳定状态的差异响应提供了对大脑中可能与年龄表型和线粒体相关疾病有关的异源线粒体景观的见解。