Age-dependent accumulation of mitochondrial tRNA mutations in mouse kidneys linked to mitochondrial kidney diseases [mtscATAC-Seq]

Heteroplasmic pathogenic mitochondrial DNA (mtDNA) mutations are known to cause mitochondrial diseases with varied clinical symptoms. The tissue-specific selection of heteroplasmic pathogenic mtDNA mutations and its correlation with clinical and pathological outcomes remains to be elucidated. To date, negative selection of pathogenic mutations has been observed in a limited range of cell types, including female germ cells, blood cells, particularly T cells, and intestinal epithelial cells. However, evidence of positive selection in any tissue or cell type has not been reported. In this study, we utilized the DddA-derived cytosine base editor (DdCBE) technique to generate a heteroplasmic pathogenic mutant mouse model harboring a mutation in the mito-tRNA Glu anticodon arm site. Over a two-year longitudinal investigation, we assessed the mutational selection dynamics in eight solid organs and blood from this model. Our findings reveal a significant positive selection for pathogenic mtDNA in the kidney and liver. Notably, the kidney exhibited mitochondrial kidney disease during aging, reflecting the clinical kidney defects observed in patients with mito-tRNA mutations. Intriguingly, disparate selection patterns emerged within multiple cell types of kidneys, with tubular epithelial cells and podocytes presenting positive selection, whereas immune cells exhibited purifying selection. Leveraging Sc-RNA seq and Stereo-seq, we identified Clu and Spp1 as molecular markers of the high mutant kidney tubular epithelial cells and suggest that up-regulation of these markers may activate the p-AKT pathway, thereby increasing the proliferation of high mutant tubular epithelial cells, hence leads to the positive selection on this site in mutant kidneys during aging. Our comprehensive analysis provides compelling evidence for the significance of tissue-specific selection of pathogenic mtDNA mutations. This study enhances our understanding of the mechanistic links between tissue-specific selection and mitochondrial disease pathogenesis, paving the way for the development of targeted therapeutic strategies in the future. Overall design: Kidneys were harvested from 3-week-old and 75-week-old mitochondrial TrnE point mutation mice and analyzed using mtscATAC-seq

异质性致病性线粒体DNA（mtDNA）突变可引发临床表现多样的线粒体疾病。目前，异质性致病性mtDNA突变的组织特异性选择及其与临床、病理结局的关联仍有待阐明。迄今仅在有限的细胞类型中观察到致病性突变的负选择，包括雌性生殖细胞、血细胞（尤其是T细胞）以及肠上皮细胞，但目前尚无任何组织或细胞类型中存在正选择的相关报道。
本研究利用DddA衍生的胞嘧啶碱基编辑器（DdCBE）技术，构建了携带有线粒体谷氨酸tRNA反密码子臂位点突变的异质性致病性突变小鼠模型。通过为期两年的纵向研究，我们对该模型的8种实体器官及血液中的突变选择动态进行了系统性评估。研究结果显示，肾脏与肝脏中存在显著的致病性mtDNA正选择。值得注意的是，肾脏在衰老进程中出现了线粒体肾病，这与线粒体tRNA突变患者所观察到的临床肾脏缺陷相一致。有趣的是，肾脏内多种细胞类型呈现出截然不同的选择模式：肾小管上皮细胞与足细胞表现为正选择，而免疫细胞则呈现纯化选择。
借助单细胞RNA测序（Sc-RNA seq）与空间转录组测序（Stereo-seq），我们鉴定出Clu与Spp1可作为高突变负荷肾脏肾小管上皮细胞的分子标志物，并提出上述标志物的上调可激活p-AKT通路，进而促进高突变肾小管上皮细胞的增殖，最终导致衰老过程中突变肾脏内该位点的正选择。
本研究的综合分析为致病性mtDNA突变的组织特异性选择的重要性提供了有力证据。本研究加深了我们对组织特异性选择与线粒体疾病发病机制之间潜在机制关联的理解，为未来开发靶向治疗策略铺平了道路。
实验总体设计：从3周龄与75周龄的线粒体TrnE点突变小鼠体内获取肾脏组织，并采用线粒体单细胞转座酶可及性染色质测序（mtscATAC-seq）进行分析。