Mitophagy-driven selection against heteroplasmic mitochondrial DNA mutations

描述(由申请人提供)：由母系遗传的致病线粒体DNA(MtDNA)突变引起的疾病可导致广泛的神经、心脏和其他疾病。不幸的是，对于这些往往具有破坏性的疾病，缺乏明显有效的临床治疗方法。一个理想的策略是消除突变的mtDNA，并用野生型(Wt)DNA取而代之。然而，经典的“基因治疗”方法很难应用于线粒体DNA突变。另一方面，线粒体经历频繁的更替(每隔几天)，甚至在有丝分裂后的细胞中也是如此，在这一过程中只复制了线粒体基因组的一部分，这为影响复制哪些mtDNA分子提供了机会。我们现在建议测试一种新的策略，以促进选择性消除有害的mtDNA突变，这种突变可以应用于异质性mtDNA突变。异质性是致病线粒体DNA突变的共同特征，指的是同一细胞或组织中wt和突变型mtDNA的混合。我们的假说利用了一种被称为“有丝分裂”(线粒体自噬降解)的自然细胞过程，这是一种选择性地消除功能障碍的线粒体的机制。我们假设，细胞内的某些线粒体将比其他线粒体具有更高水平的异质性mtDNA突变。那些有害突变水平较高的人往往会对线粒体功能造成相对较大的损害。因此，我们建议测试这一新的假设，即通过抑制携带异质性致病mtDNA突变的细胞中的mTOR激酶活性来刺激有丝分裂，将推动对突变mtDNA的选择，随着时间的推移，导致突变负担的大幅减少，从而改善线粒体的功能。我们有一个独特的资源来验证这一假设：多个SH-SY5Y胞质细胞系携带不同水平的异质性G11778A复合体I(CI)基因突变，与Leber遗传性视神经病变(LHON)相关，所有这些突变都是同时从一个家庭的成员中制备的。我们对这些细胞系的初步数据支持了我们的假设。我们实验室的第二个重要资源是表达校对缺陷mtDNA聚合酶3(Polg)的突变小鼠，导致与过早衰老表型相关的异质体细胞mtDNA突变随着年龄的增长而积累。我们的初步数据显示，这些小鼠存在显著的代谢、行为和神经化学缺陷。我们现在假设，增强Polg突变小鼠的有丝分裂吞噬作用将减少体细胞mtDNA突变的积累，并改善这些小鼠的缺陷。最终，这一策略的临床应用可能有利于与异质性mtDNA突变相关的经典线粒体疾病患者，携带与家族性帕金森综合征和其他疾病相关的Polg突变的家庭，以及潜在的与年龄相关的神经退行性疾病。 与公共卫生相关：线粒体遗传性疾病可以致残，但显然缺乏有效的治疗方法。理想情况下，人们会希望使用一种策略，消除突变的线粒体DNA，并用正常DNA取而代之。我们现在建议测试一种增强细胞消除功能障碍线粒体的自然过程的策略，我们假设这将推动对突变线粒体DNA的选择，以支持含有突变和正常DNA混合的细胞中的正常DNA。

DESCRIPTION (provided by applicant): Disorders caused by maternally inherited pathogenic mitochondrial DNA (mtDNA) mutations can lead to a wide array of neurological, cardiac, and other disorders. Unfortunately, clearly effective clinical treatments for these often devastating disorders are lacking. An ideal strategy would eliminate the mutant mtDNA and replace it with wild type (WT) DNA. However, classic "gene therapy" approaches are difficult to apply to mtDNA mutations. On the other hand, mitochondria undergo frequent turnover (every few days), even in postmitotic cells, with only a subset of copies of the mitochondrial genome being replicated during this process, providing an opportunity to influence which mtDNA molecules are replicated. We now propose to test a novel strategy to promote the selective elimination of deleterious mtDNA mutations that can be applied to heteroplasmic mtDNA mutations. Heteroplasmy is a common feature of pathogenic mtDNA mutations, and refers to a mix of WT and mutant mtDNA within the same cells or tissue. Our hypothesis takes advantage of a natural cellular process known as "mitophagy" (mitochondrial degradation by autophagy), which is a mechanism for selectively eliminating dysfunctional mitochondria. We hypothesize that some mitochondria within a cell will harbor greater levels of a heteroplasmic mtDNA mutation than others. Those with greater levels of a deleterious mutation will tend to have relatively greater impairment of mitochondrial function. Therefore, we propose to test the novel hypothesize that stimulating mitophagy by inhibiting mTOR kinase activity in cells harboring a heteroplasmic pathogenic mtDNA mutation will drive selection against the mutant mtDNA, over time leading to a substantial reduction in the mutational burden and hence an improvement in mitochondrial function. We have a unique resource available for testing this hypothesis: multiple SH-SY5Y cybrid cell lines harboring different levels of a heteroplasmic G11778A complex I (CI) gene mutation associated with Leber's Heredity Optic Neuropathy (LHON), all prepared at the same time from members of a single family. Our preliminary data with these cell lines support our hypothesis. A second important resource in our laboratory is the "mutator" mouse that expresses a proofreading deficient mtDNA polymerase 3 (Polg) leading to accumulation with age of heteroplasmic somatic mtDNA mutations in association with a premature aging phenotype. Our preliminary data demonstrate substantial metabolic, behavioral, and neurochemical deficits in these mice. We now hypothesize that enhancing mitophagy in the Polg mutator mice will attenuate the accumulation of somatic mtDNA mutations and ameliorate the deficits in these mice. Ultimately, clinical applications of this strategy have the potential to be of benefit to patients with classic mitochondrial disorders associated with heteroplasmic mtDNA mutations, to families harboring Polg mutations associated with familial parkinsonism and other disorders, and potentially for age-related neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: Mitochondrial genetic disorders can be disabling but clearly effective treatments are lacking. Ideally, one would want to use a strategy that would eliminate the mutant mitochondrial DNA and replace it with normal DNA. We now propose to test a strategy of enhancing the natural process by which cells eliminate dysfunctional mitochondria, which we hypothesize will drive selection against mutant mitochondrial DNA in favor of normal DNA in cells harboring a mix of mutant and normal DNA.