Investigation of 2‑Hydroxypropyl-β-Cyclodextrin Treatment in a Neuronal-Like Cell Model of Niemann–Pick Type C Using Quantitative Proteomics

Niemann–Pick,
type C (NPC) is a fatal, neurovisceral lysosomal
storage disorder with progressive neurodegeneration and no FDA-approved
therapy. Significant efforts have been focused on the development
of therapeutic options, and 2-hydroxypropyl-β-cyclodextrin (HP-b-CD) has emerged as a promising candidate. In cell culture,
HP-b-CD ameliorates cholesterol storage in endo/lysosomes,
a hallmark of the disorder. Furthermore, in animal studies, treatment
with HP-b-CD delays neurodegeneration and extends
lifespan. While HP-b-CD has been promising in vitro and in vivo, a clear understanding
of the mechanism(s) of action is lacking. Utilizing a neuron-like
cell culture model of SH-SY5Y differentiated cells and U18666A to
induce the NPC phenotype, we report here a large-scale mass-spectrometry-based
proteomic study to evaluate proteome changes upon treatment with these
small molecules. In this study, we show that differentiated SH-SY5Y
cells display morphological changes representative of neuronal-like
cells along with increased levels of proliferation markers. Inhibition
of the NPC cholesterol transporter 1 protein by U18666A resulted in
increased levels of known NPC markers including SCARB2/LIMP2 and LAMP2.
Finally, investigation of HP-b-CD treatment was performed
where we observe that, although HP-b-CD reduces cholesterol
storage, levels of NPC1 and NPC2 are not normalized to control levels.
This finding further supports the need for a proteostasis strategy
for NPC drug development. Moreover, proteins that were dysregulated
in the U18666A model of NPC and normalized to control levels suggest
that HP-b-CD promotes exocytosis in this neuron-like
model. Utilizing state of the art mass spectrometry analysis, these
data demonstrate newly reported changes with pharmacological perturbations
related to NPC disease and provide insight into the mechanisms of
HP-b-CD as a potential therapeutic.