# Dynamic regulation of vesicular trafficking by altered sterol homeostasis

> **NIH NIH F30** · UNIVERSITY OF SOUTH DAKOTA · 2020 · $50,520

## Abstract

PROJECT SUMMARY
A fundamental cellular process critical for normal neurodevelopment and neuronal function is clathrin-mediated
endocytosis. Within the CNS, clathrin-mediated endocytosis is functionally coupled to the exocytosis of
synaptic vesicles for neurotransmission and essential for vesicular receptor desensitization. While cholesterol
depletion is known to dramatically inhibit clathrin-mediated signaling, the specific mechanisms and
requirements underlying membrane dynamics and clathrin-mediated endocytic pathways are unknown.
Interestingly, autosomal recessive disorders of cholesterol synthesis, characterized by substitution of cellular
cholesterol for sterol intermediates, constitute a group of malformation syndromes that severely affect nervous
system development and function. Our preliminary data demonstrates clathrin-mediated endocytosis exhibits a
high degree of lipid specificity to function normally. Delineating the neurological consequences of altered sterol
homeostasis on clathrin activity will provide novel mechanistic data regarding vesicular trafficking in the context
of neurodevelopment and neuronal function. Our long-term objective for this proposal is to delineate the
cellular consequences of sterol substitution on vesicular trafficking and neuronal function. Aim 1 will utilize live-
cell imaging of CRISPR-Cas9-edited human induced pluripotent stem cells (iPSCs) to define the impact of
altered sterol homeostasis on clathrin-mediated endocytosis. Endogenous clathrin dynamics will be monitored
in real-time and quantified by live cell confocal imaging, fluorescence recovery after photobleaching (FRAP),
and total internal reflection fluorescence (TIRF) microscopy. Aim 2 will determine the molecular mechanism by
which cholesterol synthesis inhibition disrupts clathrin-mediated endocytic events through atomic force
microscopy and polarized total internal reflection fluorescence (polTIRF) microscopy to dynamically monitor
cell stiffness, membrane bending and clathrin assembly. Aim 3 will define the functional effects of sterol
substitution on vesicular trafficking and clathrin-mediated endocytosis within the neural synapse using patch-
clamp electrophysiology and high-resolution microscopic analysis of differentiated patient-derived iPSCs.
These studies will directly contribute to the understanding of clathrin-mediated endocytosis and functional
implications of disruption of this process by sterol precursors on vesicular trafficking at the mammalian
synapse. This work will provide critical evidence for disrupted clathrin-mediated trafficking underlying the
neurological deficits observed in cholesterol synthesis disorders and may support a role for dysfunction of
clathrin-mediated endocytosis in more common neurological conditions associated with altered cholesterol
levels, such as schizophrenia, Huntington's disease, and Alzheimer's disease.

## Key facts

- **NIH application ID:** 9849334
- **Project number:** 5F30NS106788-03
- **Recipient organization:** UNIVERSITY OF SOUTH DAKOTA
- **Principal Investigator:** Ruthellen Hope Anderson
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $50,520
- **Award type:** 5
- **Project period:** 2018-02-20 → 2022-02-19

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9849334

## Citation

> US National Institutes of Health, RePORTER application 9849334, Dynamic regulation of vesicular trafficking by altered sterol homeostasis (5F30NS106788-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9849334. Licensed CC0.

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