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

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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.