Mitochondrial mechanisms and vulnerability to alpha-synuclein toxicity

获得性（体细胞）线粒体DNA（mtDNA）突变随着年龄的增长而积累，并在 帕金森病（PD）早期病理阶段的多巴胺能神经元。POLG“mutator”小鼠具有一种 加速体细胞线粒体DNA突变的积累，并在 突变与PD患者神经元中发现的水平相当，表明体细胞mtDNA突变 可能在功能上很重要。此外，一些患有POLG突变的患者会出现帕金森病 与α-突触核蛋白（αSyn）病理学和黑质（SN）多巴胺能神经元丢失相关。 基于这些和其他数据，我们假设一个“两击”假说，即体细胞mtDNA突变 导致线粒体功能障碍，从而加剧对αSyn的脆弱性。这可能有助于解释 帕金森病发病率随年龄的急剧上升。本提案的总体目标是评估 体细胞mtDNA突变与α-Syn毒性之间的关系。我们将使用两种策略。第一、 我们将评估体细胞mtDNA突变水平增加对αSyn毒性的影响， 杂合和纯合POLG突变小鼠，过表达双突变体（A30 P和A53 T）αSyn， 多巴胺能神经元双突变αSyn小鼠（dMutαSyn）形成缓慢进展表型 包括伴有行为缺陷和多巴胺能SN神经元损失的纹状体多巴胺缺乏。我们 预测更早和更严重的行为缺陷，线粒体功能障碍，线粒体自噬受损， 纹状体多巴胺和多巴胺能末梢严重丢失，SN多巴胺能神经元丢失增加 与WT小鼠SN中αSyn过表达相比。第二，我们将进行立体定位SN注射， AAV-αSyn（野生型或A53 T）或野生型和杂合型的对照载体（GFP-降解决定子）， 纯合子POLG突变小鼠。我们再次预测，体细胞mtDNA突变将加剧 与αSyn表达增加相关的表型，包括神经元正常进行性死亡 在AAV-αSyn注射后观察到，导致更早和更严重的缺陷。这些研究可能会产生 第一个实验证据表明，体细胞mtDNA突变，在相关的水平在早期阶段的PD，影响 易受αSyn毒性的影响，这一结果可能部分解释了PD发病率随年龄增长而急剧上升的原因。 这些研究还将表征多巴胺能神经元变性的新小鼠模型， 结合了与PD相关的两种病理生理学机制。

Acquired (somatic) mitochondrial DNA (mtDNA) mutations accumulate with age and reach high levels in dopaminergic neurons at early pathological stages in Parkinson's disease (PD). POLG “mutator” mice have an accelerated accumulation of somatic mtDNA mutations and develop a premature aging phenotype at levels of mutations comparable to levels found in neurons in PD patients, demonstrating that somatic mtDNA mutations can be functionally significant. Furthermore, some patients with POLG mutations develop parkinsonism associated with α-synuclein (αSyn) pathology and loss of dopaminergic neurons in the substantia nigra (SN). Based on these and other data, we hypothesize a “two-hit” hypothesis whereby somatic mtDNA mutations contribute to mitochondrial dysfunction, and thereby exacerbate vulnerability to αSyn. This may help to explain the dramatic rise with age in the incidence of PD. The overall goal of this proposal is to assess the relationship between somatic mtDNA mutations and vulnerability to αSyn toxicity. We will do this using 2 strategies. First, we we will assess the impact of increased levels of somatic mtDNA mutations on αSyn toxicity by assessing heterozygous and homozygous POLG mutator mice that overexpress double-mutant (A30P and A53T) αSyn in dopaminergic neurons. Double-mutant αSyn mice (dMutαSyn) develop a slowly progressive phenotype including striatal dopamine deficiency with behavioral deficits and loss of dopaminergic SN neurons. We predict earlier and more severe behavioral deficits, mitochondrial dysfunction, impaired mitophagy, more severe loss of striatal dopamine and dopaminergic terminals, and increased SN dopaminergic neuronal loss compared to αSyn overexpression in the SN of WT mice. Second, we will perform stereotaxic SN injections of AAV-αSyn (wild-type or A53T) or a control vector (GFP-degron) in wild-type and heterozygous and homozygous POLG mutator mice. We again predict that somatic mtDNA mutations will exacerbate the phenotype associated with increased αSyn expression, including the progressive death of neurons normally seen following AAV-αSyn injections, resulting in earlier and more severe deficits. These studies may yield the first experimental evidence that somatic mtDNA mutations, at levels relevant to early stages in PD, influence vulnerability to αSyn toxicity, a result that may in part explain the dramatic rise in the incidence of PD with age. These studies also will have characterized a novel mouse model of dopaminergic neuronal degeneration that combines two mechanisms of pathophysiological relevance to PD.