# Investigating intracellular cholesterol distribution and trafficking using novel environment-sensitive cholesterol probes - supplementary instrumentation grant

> **NIH NIH R15** · FLORIDA ATLANTIC UNIVERSITY · 2023 · $96,584

## Abstract

Abstract
Cholesterol (Chol) plays a key role for neuronal 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). Cholesterol not only modifies the trafficking and cleavage of amyloid
precursor protein (APP) but also worsens the aggregation of β-amyloid peptides (Aβs) and Tau protein.
Moreover, abnormality of membrane-embedded cholesterol 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 cholesterol distribution than the global regulation of cholesterol metabolism. Studying
cholesterol transport, especially in live cells, requires fluorescent probes and imaging capability that can not only
mimic membrane cholesterol 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 use 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 different properties. 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 inhibitors, we will ask how the secretase cleavages of APP affect mChol. Furthermore, we will
ask how such altered mChol regulation associates with 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
an interdisciplinary research experience.

## Key facts

- **NIH application ID:** 10796611
- **Project number:** 3R15GM147912-01S1
- **Recipient organization:** FLORIDA ATLANTIC UNIVERSITY
- **Principal Investigator:** Maciej J. Stawikowski
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $96,584
- **Award type:** 3
- **Project period:** 2022-09-01 → 2025-08-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10796611

## Citation

> US National Institutes of Health, RePORTER application 10796611, Investigating intracellular cholesterol distribution and trafficking using novel environment-sensitive cholesterol probes - supplementary instrumentation grant (3R15GM147912-01S1). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10796611. Licensed CC0.

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