Abstract
Cholesterol (Chol) plays a key role for cell functionality and survival, the disruption of cellular Chol
homeostasis has been linked to major neurodegenerative disorders including Parkinson's disease,
Huntington's disease, and Alzheimer's disease (AD). Chol not only modifies the trafficking and cleavage of
amyloid precursor protein (APP) but also worsens the aggregation of ß-amyloid peptides (Aßs) and Tau
protein. Moreover, abnormality of membrane-embedded Chol can cause synaptic dysfunction by impairing
the turnover of synaptic vesicles (SVs) and the activities of postsynaptic receptors. While the vast majority
studies have been focused on Chol's cellular metabolism, more and more reports have suggested that
intracellular Chol transport via membrane trafficking or newly discovered sterol transporters provides more
local control of subcellular membrane Chol distribution than the global regulation of Chol metabolism. Studying
Chol transport, especially in live cells, requires fluorescent probes that can not only mimic Chol but also
distinguish the plasma membrane and intracellular compartments like endosomes. In this project we propose
to develop a class of environment-sensitive fluorescent Chol probes (CNDs) using 1,8-naphthalimide
fluorophore and employ them to elucidate the subcellular distribution and trafficking of Chol as well as its
implication in AD pathogenesis. We will take a multidisciplinary strategy with advanced technologies including
computational chemistry, chemical synthesis and live-cell imaging. Based on the modular scaffold of our
proposed probes, we will generate new CNDs with distinct properties ideal for different applications. We will
determine the uptake, distribution, and dispersion of selected CNDs in different cell types (3T3 fibroblast cells,
astrocytes, and neurons) and different subcellular compartments (e.g., different organelles including
endosomes, lysosomes and lipid droplets) with high spatiotemporal precision. To study CNDs' resemblance to
endogenous Chol, we will ask how disrupting cellular processes and activity of proteins involved in Chol
uptake, intracellular transport, sequestration, and dispersion will affect CNDs staining. Combining selected
CNDs with spectrally separable reporters for synaptic vesicles (SVs) and APP, we will study how Chol in
different membrane compartments affect SV turnover, APP distribution and cleavage in major types of brain
cells. Using transgenic methods, we will ask how APP and its major cleavage product affect mChol trafficking
and homeostasis. Using pharmacological tools, we will ask how the secretase cleavages of APP affect mChol.
Furthermore, we will address how such altered mChol regulation contribute to synaptic dysfunction and
neuronal loss commonly found in AD. In summary, this project will rejuvenate the studies of membrane Chol
and give the participating students a cross-disciplinary research experience.
