Mitophagy-driven selection against heteroplasmic mitochondrial DNA mutations

描述（由申请人提供）：由母系遗传致病性线粒体DNA（mtDNA）突变引起的疾病可导致广泛的神经系统、心脏和其他疾病。不幸的是，缺乏对这些通常具有破坏性的疾病的明确有效的临床治疗。理想的策略是消除突变的mtDNA并用野生型（WT）DNA取代它。然而，经典的“基因治疗”方法很难应用于mtDNA突变。另一方面，线粒体经历频繁的周转（每隔几天），即使在有丝分裂后的细胞中，在此过程中只有线粒体基因组拷贝的一个子集被复制，这提供了一个影响mtDNA分子复制的机会。我们现在提出测试一种新的策略，以促进选择性消除有害的mtDNA突变，可以应用于异质性mtDNA突变。异质性是致病性mtDNA突变的共同特征，是指WT和突变mtDNA在同一细胞或组织中的混合。我们的假设利用了一种被称为“线粒体自噬”（线粒体自噬降解）的自然细胞过程，这是一种选择性消除功能失调的线粒体的机制。我们假设细胞内的某些线粒体比其他线粒体具有更高水平的异质性mtDNA突变。那些具有更高水平的有害突变的人将倾向于具有相对更大的线粒体功能损伤。因此，我们建议测试新的假设，即通过抑制具有异质性致病性mtDNA突变的细胞中的mTOR激酶活性来刺激线粒体自噬，将驱动对突变mtDNA的选择，随着时间的推移，导致突变负荷的大幅减少，从而改善线粒体功能。我们有一个独特的资源可用于测试这一假设：多个SH-SY 5 Y胞质杂交细胞系含有不同水平的异质性G11778 A复合物I（CI）基因突变与Leber遗传性视神经病变（LHON），所有准备在同一时间从一个家庭的成员。我们对这些细胞系的初步数据支持我们的假设。我们实验室的第二个重要资源是“突变”小鼠，其表达校对缺陷型mtDNA聚合酶3（Polg），导致与过早衰老表型相关的异质性体细胞mtDNA突变随年龄积累。我们的初步数据表明，这些小鼠存在大量的代谢、行为和神经化学缺陷。我们现在假设，增强Polg突变小鼠的线粒体自噬将减弱体细胞mtDNA突变的积累，并改善这些小鼠的缺陷。最终，这种策略的临床应用有可能对与异质性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突变的家族以及可能对年龄相关的神经退行性疾病患者有益。 公共卫生相关性：线粒体遗传疾病可能导致残疾，但缺乏明显有效的治疗方法。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，并用正常的DNA取代它。我们现在建议测试一种增强细胞消除功能失调的线粒体的自然过程的策略，我们假设这将在含有突变和正常DNA混合的细胞中驱动对突变线粒体DNA的选择，而有利于正常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.