Neurodegenerative diseases represent a significant and increasing burden on society. In particular, Alzheimer's disease and Parkinson's disease are the most common neurodegenerative disorders in the world and affect 5.4 million and >500,000 people in the USA, respectively. In fact, the global cost of Alzheimer's disease and related dementias was estimated in 2010 to be ~1% of the world's gross domestic product (GDP). Despite the knowledge that virtually all neurodegenerative diseases originate with problems in the folding and trafficking of specific proteins in cells, there are simply no effectiv treatments to halt or cure these diseases. This is at least in part due to the lack of a mechanisti understanding of the disease pathology at the cellular level. The research proposed herein uses the budding yeast, Saccharomyces cerevisiae, as a cellular model system. This system has previously been shown to recapitulate unique features of neuronal toxicity observed in human neurodegenerative diseases. Significantly, all previous analyses in both yeast and neuronal models were limited to steady-state bulk measurements, which average over many cells and mask essentially all intracellular dynamics. These measurements cannot reveal important dynamic properties, which require observation of single cells over time. By contrast, proposed herein is a novel approach to systematically measure the global response of single cells to the expression of disease-relevant proteins, such as ?-synuclein (Parkinson's disease) and the A? peptide (Alzheimer's disease) by following the time-dependent changes of biochemical pathways, protein localization and trafficking, organelles, and metabolites in single cells. Biological circuits function inside cells and hence require a single-cell analysis to uncover all their behaviors. The single-cell resolution will be achieved through the development of an innovative microfluidic analysis platform, which permits the imaging of individual cells over time and allows for the quantification of dynamic responses in single cells. Such systematic single-cell measurements will reveal novel mechanisms relevant to cellular toxicity and will contribute to a systems-level understanding of how A? and ?-synuclein poison cellular physiology. Further, this high-throughput imaging platform will be employed to investigate the cell biological effects of ~100 small- molecule compounds that were previously identified through large-scale small-molecule screening as potential drug candidates and are currently being verified for their efficacy in neurons. Discoveries made with the high- throughput imaging platform described herein will later be validated in mammalian neuronal systems. Significantly, the systems-level, single-cell analysis approach described herein has never before been applied to the investigation of neurodegenerative diseases and has the potential to uncover novel insights about intracellular perturbations on a global scale, which is necessary for identifying effective therapeutics to combat these diseases.

Public Health Relevance

Alzheimer's disease and Parkinson's disease are incurable neurodegenerative disorders that lack efficacious treatments due to the absence of a mechanistic understanding of their underlying pathologies, although the initiating steps are known to involve intracellular protein misfolding and protein aggregation. Our proposed research combines single-cell time-lapse microscopy and microfluidics to investigate how the disease-relevant A? peptide (Alzheimer's disease) and ?-synuclein protein (Parkinson's disease) globally poison live cells and to obtain a systems-level understanding of the perturbations. We will also apply this single-cell analysis platform to assess a select number of small-molecule compounds, which were recently identified in a large-scale high-throughput screen and are currently being validated in neurons, to elucidate their mechanisms of action;this may lead to the identification of novel effective therapeutics.

National Institute of Health (NIH)
Exploratory/Developmental Grants (R21)
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Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
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Sutherland, Margaret L
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Whitehead Institute for Biomedical Research
United States
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