Towards Improved Early Diagnosis of Neurodegenerative Diseases - Noninvasive Investigations of Retinal Metabolism Using Fluorescence Lifetime and Anisotropy Analysis of the Cellular Redox State

神经退行性疾病（ND）对日益老龄化的人口构成巨大的医疗和社会经济挑战。尽管它们的病因多样，但导致细胞功能障碍和死亡的神经元病理生理似乎有共同的下游途径。由于线粒体提供大部分细胞ATP并调节细胞凋亡，它在细胞死亡和神经退行性变的主要原因之间建立了中心联系。此外，线粒体功能障碍（MD）越来越多地被认为是主要ND（如阿尔茨海默病等）发病机制的关键步骤，也是糖尿病视网膜病变（DR）、年龄相关性黄斑变性（AMD）和青光眼等眼科疾病发病机制的关键步骤。MD的早期诊断可能有助于建立和改进治疗方法，但更重要的是扩展目前对神经变性初始过程的理解。因此，检测线粒体能量代谢的初始凋亡前变化将是最大的兴趣。眼底自体荧光（FAF）成像作为一种新兴的眼科诊断技术，可以有效地揭示病理改变的重要细节。我们开发了一种新的无创光学成像技术，称为荧光寿命成像眼科检查（FLIO），它是基于光谱分辨荧光寿命。与稳态荧光相反，FLIO可以精确测量内源性荧光团微环境的变化。因此，它可以探测辅酶NAD(P)H和黄素的氧化还原平衡，以及检测部分毒性代谢副产物。使用FLIO进行的首次临床研究显示，糖尿病患者、amd患者和对照组之间存在显著差异。然而，体内测量的解释需要更深入地了解FAF的病理改变。因此，本项目的主要目标是建立视网膜自身荧光病理变化与初始凋亡前MDs之间的详细联系。为了研究视网膜变性（即DR和AMD）的发病机制，将采用非侵入性光学技术和各种生化细胞测定。特别是，光谱和时间分辨荧光以及荧光各向异性将用于探测细胞和器官培养模型中对缺氧、高血糖和光氧化应激的反应的游离和蛋白质结合的NADH和黄素的浓度。为此目的，将开发一种新的记录技术以及分析荧光数据的新模型和算法。总之，这可能为DR和AMD的早期诊断提供了巨大的进步，从而为影响数百万患者的视网膜变性提供了新的治疗策略。此外，我们的研究将大大有助于对ND发展的基本机制的基本理解。

Neurodegenerative diseases (ND) constitute a dramatic medical and socio-economic challenge for the increasingly ageing population. Despite their diverse aetiology, neuronal pathophysiology leading to cell dysfunction and death seems to have common downstream pathways. As the mitochondrion provides most of cellular ATP and regulates apoptosis, it creates a central link between cell death and the primary cause of neurodegeneration. Moreover, mitochondrial dysfunction (MD) has been increasingly implicated as a crucial step in the pathogenesis of not only major ND (e.g., Alzheimer's disease, etc.), but also ophthalmological disorders such as diabetic retinopathy (DR), age-related macular degeneration (AMD) and glaucoma. Early diagnosis of MD might help to establish and improve therapeutic approaches, but more importantly extend the current understanding of the initial processes of neurodegeneration. Therefore, detecting initial pre-apoptotic changes in the energy metabolism of mitochondria would be of utmost interest.As an emerging technique in ophthalmological diagnostics, ocular fundus autofluorescence (FAF) imaging can be a powerful technique to reveal important details on pathological alterations. We developed a new noninvasive optical imaging technique called fluorescence lifetime imaging ophthalmoscopy (FLIO), which is based on spectral-resolved fluorescence lifetime. As opposed to steady state fluorescence, FLIO allows accurate measurement of changes in the microenvironment of endogenous fluorophores. Thereby, it enables probing of redox equilibria of the coenzymes NAD(P)H and flavins as well as the detection of partially toxic metabolic by-products. First clinical studies using FLIO revealed a significant discrimination of diabetic patients, AMD-patients and controls. However, the interpretation of in vivo measurements requires deeper insights into the pathological alterations of FAF.Therefore, the main goal of this project is to establish a detailed connection between pathological changes of retinal autofluorescence and initial pre-apoptotic MDs. To investigate the pathogenesis of retinal degeneration (i.e. DR and AMD) noninvasive optical techniques and a variety of biochemical cell assays will be employed. Particularly, spectral- and time-resolved fluorescence as well as fluorescence anisotropy will be used to probe the concentrations of free and protein-bound NADH and flavins in cell and organ culture models in response to hypoxy, hyperglycemia and photooxidative stress. For that purpose, a new recording technique as well as new models and algorithms for the analysis of fluorescence data will be developed. Altogether, this may provide great advances in early diagnosis of DR and AMD, and thus will support new therapeutic strategies for retinal degeneration affecting millions of patients. Moreover, our investigations will considerably contribute to a fundamental understanding of essential mechanisms involved in the development of ND.