MT Function and Dysfunction in Single Neurons in Vivo

描述(由申请人提供)：本申请涉及广泛的挑战领域(06)：使能技术，和具体的挑战主题，01-OD-101：开发新的工具和技术来询问活体中的人类线粒体功能。线粒体功能障碍(MT)被认为是衰老和神经退行性疾病的主要因素。尽管MT的功能可以通过氧化磷酸化和ATP产生的公认特性来衡量，但这些明确的衡量标准并没有直接导致对病理生理学的明显理解。阿尔茨海默氏症(AD)、帕金森氏症(PD)、亨廷顿氏症都有MT跟不上细胞的能量需求的特点。然而，AD、PD和HD显示特定区域的细胞死亡，即使在这些区域内，也只针对特定类型的细胞。因此，地区性特异性意味着MT功能障碍的发生是对特定细胞中随年龄或疾病进展而变化的细胞代谢的反应。由于系统的异质性，存在大量的障碍，使得很难确定主要的缺陷是特定的MT不能将代谢前体转化为ATP，还是细胞途径不能产生足够的代谢前体来合成ATP。为了克服这些限制，我们开发了一种全新的、超高灵敏度的质谱学技术，称为纳米结构引发质谱学(NIMS)，它能够在150 nm分辨率下进行质量分析。因此，NIMS具有前所未有的能力来空间跟踪单个细胞或单个MT中的质量组成。NIMS的设计目的是创建分子特征，即使是相邻的细胞类型也能唯一区分，并且不会破坏大脑的三维结构。我们已经使NIMS能够根据单个细胞在整个脑片内任何区域的代谢物组成对其进行成像和定位，并在原位直接测量单个细胞中MT特异性的变化。碎片、灵敏度和低空间分辨率的问题限制了其他质谱学技术的使用，以实现NIMS的分辨率和成像能力。然而，我们的方法提供了可能是唯一有效和精确地解决区域、细胞类型和MT特异性异质性的现有技术进步。NIMS可以应用于任何疾病，任何组织切片，并被认为是分类大脑中发生的复杂变化的想法。 与公共健康相关：至少2%的美国人将患有某种形式的阿尔茨海默病(AD)(400万人)、帕金森氏病(PD)(150万人)或亨廷顿病(HD)(20万人)等。这些疾病中的每一种都可以影响患者数十年，但目前还没有有效的长期治疗方法。由于受影响的人数将呈指数级增长，问题的规模与我们的治疗能力之间的差距将会扩大。因此，了解MT功能障碍的关键作用有望增加治疗选择。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06): Enabling Technologies, and specific Challenge Topic, 01-OD-101: Development of new tools and technologies to interrogate human mitochondrial function in vivo. Dysfunction of Mitochondria (MT) dysfunction is thought to be a primary contributor to aging and neurodegenerative disease. Although MT function is measurable by well-established properties of oxidative phosphorylation and ATP production, these defined measures have not directly led to an obvious understanding of pathophysiology. Alzheimer's (AD) Parkinson's (PD) Huntington's disease shares the property that MT is not keeping up with the energy demands of the cell. However, AD, PD, and HD display region-specific cell death, and even within those regions, only select cell types are targeted. Regional specificity, therefore, implies that MT dysfunction develops in response to the changing cellular metabolism in only specific cells with age or as disease progresses. Due to system heterogeneity, there have been substantial barriers that have made it difficult to determine whether the primary defect is the inability of particular MT to convert metabolic precursors to ATP, or a failure of cellular pathways to generate sufficient metabolic precursors for ATP synthesis. To overcome these limitations, we have developed a completely new, ultra-high sensitivity mass spectrometry technique called Nanostructure-Initiator Mass Spectrometry (NIMS), which is capable of mass analysis at 150nm resolution. Thus, NIMS has the unprecedented ability to spatially track mass composition in a single cell or in a single MT. NIMS is designed to create molecule signature that uniquely distinguish among even adjacent cell types, and does not destroy the 3-dimenational architecture of the brain. We have adapted NIMS to image and localize individual cells by their metabolite composition in any region within whole brain slices, and to directly measure MT-specific variations in single cells in situ. Issues with fragmentation, sensitivity, and low spatial resolution have limited the use of other mass spectroscopy techniques to achieve the resolution and imaging capabilities NIMS. However, our approach provides, perhaps the only existing technological advance that efficiently and precisely resolve, region, cell type, and MT-specific heterogeneity. NIMS can be applied to any disease, to any tissue section, and is idea to sort out complex changes that occur in the brain. PUBLIC HEALTH RELEVANCE: At least 2% of Americans will be afflicted with some form of Alzheimer's disease (AD) (4,000,000), Parkinson's disease (PD) (1,500,000), or Huntington's disease (HD) (200,000), among others. Each of these disorders can affect patients for decades, yet no effective long-term approaches to therapy are currently available. Because the number of affected individuals will grow exponentially, the gap between the problem's size and our capabilities for treatment will widen. Thus, an understanding of the pivotal role of MT dysfunction promises to increase therapeutic options.