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. Author manuscript; available in PMC: 2025 May 24.
Published in final edited form as: J Am Soc Mass Spectrom. 2023 Mar 15;34(4):668–675. doi: 10.1021/jasms.2c00342

Investigation of 2-hydroxypropyl-beta-cyclodextrin treatment in a neuronal-like cell model of Niemann-Pick type C using quantitative proteomics

Antony Cougnoux 1, Melissa R Pergande 2, Fidel Serna-Perez 2, Stephanie M Cologna 2
PMCID: PMC12102752  NIHMSID: NIHMS2080134  PMID: 36920149

Abstract

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-beta-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 are 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.

Introduction

Niemann-Pick, type C (NPC) is a rare, autosomal recessive disorder with currently no FDA-approved treatment. The disease is caused by mutation in the gene NPC1 (95% of cases) or NPC2 (5% of cases)1 and the loss or reduced function of either protein leads to the abnormal accumulation of unesterified cholesterol and glycosphingolipids in the endosomal-lysosomal system in every cell. As a result of this lipid storage burden, neurological deficits, including ataxia, cataplexy, seizures dementia, and ultimately premature death occur.1 Additionally, peripheral disease exists including notably hepatosplenomegaly and other organ dysfunction. To address the severe nature of NPC, several candidate treatments are being investigated with 2-hydroxypropyl-beta-cyclodextrin (HP-b-CD) among the most promising where both cell culture and animal studies have proven a reduction in lipid storage.2,3 In both small and large animal models, HP-b-CD has significantly delayed the course of the disease,3–5 slowing neurodegeneration and extending lifespan. The favorable results seen in NPC models has led to ongoing clinical evaluation.6,7

Cyclodextrins are cyclic oligosaccharides that can accommodate binding of hydrophobic molecules, such as cholesterol, without hindering their solubility properties. Efforts to better understand the mechanism of HP-b-CD in the context of NPC have revealed crucial findings and suggest that HP-b-CD may be endocytosed,8 induces lysosomal exocytosis,9 and promotes endo/lysosomal secretion in non-specialized cells10. Moreover, recent work suggests that HP-b-CD promotes lipid redistribution at the plasma membrane rather than exocytosis or efflux.11. Focusing on the central nervous system, Barthelemy et al., showed that HP-b-CD treatment in retinal neurons utilizes lamellar inclusions and activity of glial cells further revealing the complexity of this potential treatment and the possibility that multiple mechanisms are present, and this can differ by cell type.12

While HP-b-CD has been found to be safe and is currently used in patients, the exact mechanism(s) of action is not fully understood. Furthermore, proteomic studies following HP-b-CD treatment in NPC models has not been performed. To fill this important gap of knowledge, we leveraged high resolution/high mass accuracy mass spectrometry to investigate the proteomic changes induced by HP-b-CD treatment in a neuronal cell model of NPC. To this end, an extensive breadth of literature has shown that NPC can be mimicked in vitro by treating cells with the class II amphiphile drug U18666A.13 To model neuronal cells, we used the SH-SY5Y human neuroblastoma cells, which can be differentiated into neuron-like cells following retinoic acid treatment14. These cells have extensively served as tools to investigate neurodegenerative processes, such as Alzheimer disease and neurotoxicity, and offers several advantages (e.g., human origin, reproducible neuronal-like differentiation, etc.). Herein, we report protein changes associated with retinoic acid differentiation, U18666A treatment to inhibit cholesterol efflux mimicking NPC, and finally changes to the proteome associated with HP-b-CD treatment.

Methods.

Reagents

SH-SY5Y human neuroblastoma cells were obtained from American Type Culture Collection (CRL-2266, ATCC, Manassas, VA). 2-hydroxypropyl-beta-cyclodextrin was from Roquette (Kleptose HPB, Roquette, Vacquemont, France), U18666A was from Sigma (St. Louis, MO, USA). Trans-retinoic acid (RA) was purchased from Acros/Fisher Scientific (Hampton, NH, USA). Cell culture media, antibiotics and enzymes were procured from Life Technologies (Carlsbad, CA, USA) and LC-MS grade solvents were received from Sigma. All reagents and chemicals were used as received unless otherwise noted.

Cell Culture and Differentiation

SH-SY5Y human neuroblastoma cells were grown in medium (1:1 mixture of Ham’s F-12 medium and modified Eagle’s medium) supplemented with 10% heat inactivated fetal calf serum and penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA) at 37°C in a humidified atmosphere with 5% CO2. Neuronal differentiation was induced by the addition of trans-retinoic acid14,15 at 10μM for 48hours. Following the differentiation, the cells were split into 4 groups: untreated, 2.5μg/mL U18666A treated, HP-b-CD treated and U18666A & HP-b-CD treated and maintained for 72 hours in these condition prior to analysis (A schematic description is provided as Figure S1).

Sample preparation, iTRAQ labeling and fractionation

Cells were detached via trypsinization, washed and pelleted. Cell pellets for the undifferentiated (n=4) cultures as well as the differentiated untreated (n=3), U18666A treated (n=3), HP-b-CD treated (n=3) and U18666A & HP-b-CD treated (n=3) conditions were lysed in 5% w/v sodium dodecyl sulfate and protein concentration determined using a bicinchoninic assay. Fifty micrograms of protein for each sample were aliquoted into a new Eppendorf tube, spiked with green fluorescent protein (trypsin digestion quality control standard) at a concentration of 50fmol per 1μg protein and processed via the S-trap protocol (Protifi) using triethylammonium bicarbonate buffer. Tryptic peptides were labeled using two 8-plex iTRAQ Reagent kit (AB Sciex, Framingham, MA), according to manufacturer instructions (Kit 1 labeling: 113-undifferentiated, 114-U18666A, 115-U18666A, 116-untreated, 117-U18666A, 118-undifferentiated, 119-untreated, 121-untreated; Kit 2 labeling: 113- U18666A & HP-b-CD, 114-undifferentiated, 115-HP-b-CD, 116- U18666A & HP-b-CD, 117-undifferentiated, 118-HP-b-CD, 119-HP-b-CD, 121- U18666A & HP-b-CD). Labeled samples were combined per kit, lyophilized, resuspended in 0.1% formic acid, and purified using strong cation exchange chromatography (PolyLC, Columbia, MD). The resulting labeled peptides were fractionated (20 fractions per kit) via high pH reversed-phase chromatography similar to that previously described.16 In brief, a Waters XBridge C18 3.5μm 4.6×250mm column was used to separate peptides under basic (pH) conditions at a flow rate of 1mL per minute. Fractions were collected each minute over a 60-minute gradient, then concatenated into 20 fractions. Each combined fraction was then lyophilized and resuspended in 20 μL 0.1% formic acid prior to nano-LC-MS/MS analysis.

Nano-LC-MS/MS analysis and data analysis

Mass detection occurred with an Orbitrap Q-Exactive mass spectrometer (ThermoFisher Scientific) equipped with an Agilent HPLC system. Chromatographic separation of peptides was accomplished using a Zorbax 300SB-C18 column (3.5 μm ID × 150 mm, particle size 5 μm, pore size 100 Å, Agilent Technologies). The peptides were loaded onto a Zorbax 300SB-C18 trap cartridge at a flow rate of 3 μL/minute for 10 minutes. After washing with 0.1% formic acid, the peptides were eluted using a 5–40% B gradient for 60 minutes at a flow rate of 250 nL/minute acid (mobile phase A =01% formic acid; mobile phase B = 0.1% formic acid in acetonitrile). The flow-through was analyzed using data-dependent analysis settings to select the top 10 most abundant ions for higher energy collisional dissociation fragmentation. Proteins were identified by searching MS/MS spectra against the Human SwissProt database using the Sequest search engine in the Proteome Discoverer software employing a 1% false discovery rate (FDR). Here, trypsin was set as the protease with two missed cleavages and searches were performed with precursor and fragment mass error tolerances were set at 10 ppm and 0.1 Da, respectively. Peptides precursors of +2, +3 and +4 were considered. Variable modifications were set to iTRAQ8(Y), oxidation (M) and deamination (NQ), whereas carbamidomethyl (C) and iTRAQ8(K)(N-term) were set as fixed modifications. Protein search results were imported into Scaffold Q+ where protein identifications were accepted at 99.0% probability and contained at least 2 identified peptides. Here, both iTRAQ data sets were combined using the undifferentiated SH-SY5Y samples. The resulting processed data was imported into Mass Profiler Professional (Agilent Technologies, Santa Clara, CA, USA) where fold changes were determined and one-way ANOVA (p < 0.05) performed. Mass spectrometry proteomics data have been deposited to the MassIVE repository with the dataset identifier MSV000087053.

Computational Analysis

All plots were generated in ggplot217 using R 4.2.1 and Rtools 4.2 in the R programming language. Pathway analysis was performed using Gprofiler218 (user_threshold = 0.05, correction_method = “fdr”, domain_scope = “annotated”), STRING.19 Pathways from the pathway analysis outputs were manually classified into: “Exocytosis”, “Immunity”, “Lysosome”, “Metabolism”, “Sterol/Lipid”, “Hypoxia” and “Other”.

Results

The rapid development in mass spectrometry instrumentation and robust separation platforms have enabled large scale proteomic measurements to reveal insights to biological processes including those associated with human disease. In the current study, we carried out a differential proteomic study using a human neuronal-like cell culture model to investigate the proteomic changes associated in HP-b-CD treatment, a candidate drug that is currently being evaluated for the treatment of NPC disease. To do so, we implemented isobaric tagging using a pooled reference approach allowing for comparisons across multiple conditions and inclusion of biological replicates. The use of the SH-SY5Y cell line can represent a neuronal-like model with induced differentiation by retinoic acid. Therefore, we first evaluated the effects of retinoic acid and evaluated neuronal markers. Next, treatment with U18666A, an inhibitor of the NPC1 protein, was used to induce endo/lysosomal cholesterol storage, mimicking NPC disease. Finally, to determine the proteome changes associated with HP-b-CD and to gain further mechanistic insight we evaluated how protein levels responded to HP-b-CD treatment in the above-mentioned cell model.

Mass spectrometry analysis supports the cellular differentiation following retinoic acid treatments.

SH-SY5Y cells treated for 48h with 10uM of trans-retinoic acid acquired different morphological features compared to untreated cells. The cells become elongated with the development of thin extensions similar to axon or dendrite characteristics of neurons (Figure 1A). These morphological changes are in accordance with prior reports and the lower cell density is due to the lack of proliferation of the differentiated cells which is well documented in the literature.14,20,21 To support the visual inspection of this differentiation, we performed a LC-MS/MS proteomic analysis using isobaric tagging on retinoic acid treated and untreated SH-SY5Y cells. From the data, we manually extracted the expression levels of neuronal markers INA (alpha-internexin), NES (nestin), NEFM (neurofilament medium polypeptide) and NEFL (neurofilament light polypeptide) as well as the proliferation marker PCNA (proliferating cell nuclear antigen) (Figure 1B). As expected, the proliferation maker was decreased while the neuronal markers were increased following treatment.

Figure 1: Characterization of SH-SY5Y cells differentiation.

Figure 1:

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A) Representative bright field image of SH-SY5Y untreated (undifferentiated) and treated for 48h with 10 μM of retinoic acid (differentiated). *p < 0.05. B) Box plots of selected neuronal and proliferation markers (log2 normalized expression), where undifferentiated cells are displayed in black (N=4), and retinoic acid treated in gray (N=3). C) Representative pathway increased following retinoic acid treatment. D) Representative pathways decreased following retinoic acid treatment. (Complete list provided in Table S1).

To gain further insights to the differentiation of this widely used in vitro model, we performed pathway analysis on the 697 proteins significantly altered proteins (p < 0.05), where 212 were observed to be increased and 485 decreased (Table S1) in the differentiated cultures relative to the untreated cultures. In our analysis of overexpressed proteins, cytoskeleton remodeling, axon and synapses organization were significantly enriched supporting the differentiation process (Figure 1C, Table S1). Pathway analysis on the down regulated proteins unsurprisingly revealed a reduction in the mechanisms associated with cell division including: mitosis, cell cycle progression and DNA replication (Figure 1D, Table S1) as previously described by others.22,23 Altogether the proteomic results and the subsequent analysis confirms the extensive literature of retinoic acid induced changes in SH-SY5Y cells, differentiating them into neurons-like cells.

U18666A induces a NPC1-like phenotype in the differentiated SH-SY5Y cells.

Following the validation of the neuronal features of the cells, we investigated if 48h of U18666A treatment at 2.5ug/mL induced the NPC1-like phenotype in these cells as has been described by others.15 The hallmark of this lysosomal storage disease is the endo/lysosome accumulation of unesterified cholesterol and other glycolipids.1 To validate the NPC phenotype, we extracted from the proteomic data proteins known to be over expressed from genetic models in NPC including: DHCR24,24 LAMP2,25 and SCARB2.26 The analysis on U18666A treated SH-SY5Y cells supports the previous observations (Figure 2A, Table S2).

Figure 2: Characterization of differentiated SH-SY5Y cells treated with U18666A.

Figure 2:

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A) Box plot of protein markers known to be altered in NPC (Untreated in black (N=3), U18666A treated in gray (N=3); * = p<0.05. B) Distribution of the altered pathways from U18666A treatment that are decreased or increased. C) Representative pathways enriched following analysis on the proteins increased by U18666A treatment. (Complete list provided in Table S2).

Further investigation of the mass spectrometry data revealed 89 proteins to be significantly altered with U18666A treatment: 59 increased and 30 decreased compared to control. The downstream pathway analysis on these proteins agrees with the known biology of the disease, where many of the pathways significantly enriched are linked to sterol/lipid metabolism (60%) and lysosome function (16%) (Figure 2B, Table S2) with cholesterol synthesis pathways being the most enriched (Figure 2C, Table S2). Metabolic pathways not related to lipids account for 14% of the changes. Immune related pathways account for 3%. Pathways labelled as “Other” include pathways such as “cell activation” and “phosphoprotein”.

2-hydroxypropyl-beta-cyclodextrin has a broad effect on the proteome.

β-cyclodextrins, and notably HP-b-CD are hydrophobic molecule carriers known to efficiently bind cholesterol.27 By doing so, a plethora of receptors and their downstream signal transducers can be affected in cells treated with compounds belonging to this family.27,28 HP-b-CD promotes the redistribution of lipids in the cellular,11 as a consequence, membrane bound receptors, and their downstream signaling, may be affected by the treatment. We first extracted from the proteomic data the proteins that are known to be over expressed in NPC as previously reported including: DHCR24, LAMP2 and SCARB2. With the exception of DHCR24, a component of the cholesterol biosynthesis pathway, the other proteins were increased following HP-b-CD treatment, independently of the NPC phenotype (Figure 3A).

Figure 3: Characterization of differentiated SH-SY5Y cells treated with HP-b-CD.

Figure 3:

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A) Box plot of the proteins known to be induced by U18666A following HP-b-CD treatment. Untreated in black (N=3), HP-b-CD treated in gray (N=3) * p<0.05. B) Venn diagram of the proteins overexpressed in cells treated with U18666A and HP-b-CD found in independent experiments. C) Distribution of pathways that decreased or increased by HP-b-CD treatment. (Complete list provided in Table S2). D) Representative pathways enriched following HP-b-CD treatment of neuronal-like cells (Complete list provided in Table S2).

Next, we evaluated the broad proteomic changes with HP-b-CD treatment. From this analysis 245 proteins were significantly altered: 172 increased and 73 decreased compared to control (Table S2). A strong overlap between both treatments at the proteomic level was observed. We observed 29 of the proteins increased by U18666A are also by HP-b-CD (Figure 3B). This observation agrees with our hypothesis that HP-b-CD has a broad action on healthy cells potentially affecting numerous pathways. In our analysis 89 cellular pathways were affected by HP-b-CD treatment (37 decreased and 52 increased, Table S2) whereas U18666A treated cells only had 59 pathways significantly affected (Table S2). In addition, the breadth of molecular mechanism modified by HP-b-CD is broader than U18666A. Indeed, an enrichment in pathways related to exocytosis (6% of the increased pathways) and hypoxia (2% of the increased pathways) increased in the HP-b-CD treated cells appears specific to this group (Figure 3C–D).

Cells treated with molecules such as cyclodextrins are stripped from their membrane cholesterol27 and therefore, we expected to observe a cholesterol deficient signature which surprisingly was not found. However, we measured an increased in NPC1 (Log2FC = 4, p = 0.003) and NPC2 (Log2FC = 0.92, p = 0.03) proteins following HP-b-CD treatment suggesting an improved recruitment or efflux of the unesterified cholesterol stored in the endo/lysosome. These two proteins are pivotal in the recycling of unesterified cholesterol through the endo/lysosome and therefore may participate the HP-b-CD induced lipid redistribution.9,11

2-hydroxypropyl-beta-cyclodextrin treatment abolishes U18666A induced changes.

Thus far, we have shown that differentiated SH-SY5Y cells acquired neuronal features and these neuron-like cells, once treated with U18666A, mimic NPC. We next evaluated the effect of HP-b-CD proteome wide in this NPC model. We began our evaluation by extracting from the proteomic data proteins expected to be elevated upon U18666A treatment (Figure 2) and in NPC including DHCR2424, LAMP225 and SCARB226 (Figure 4A). Additionally, we identified a significant increase in NPC1 and NPC2 proteins following HP-b-CD treatment (Figure 4A). Elevation of these proteins has been considered therapeutic avenue in NPC.29 From this subset of proteins, when our NPC neuronal-like cells (U18666A treated cells) are treated with HP-b-CD, we measured a decreased in DHCR24 and NPC2 toward the level of untreated or HP-b-CD when compared to U18666A (Figure 4A). On the contrary, in the U18666A & HP-b-CD group, we measured increased levels of NPC1 and SCARB2 toward the level of HP-b-CD only treated cells when compared to the control and U18666A groups (Figure 4A). LAMP2 was increased to similar levels in all three conditions compared to control cells.

Figure 4: Characterization of neuronal-like cells with an NPC phenotype treated with HP-b-CD.

Figure 4:

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A) Box plot of the proteins known to be altered in NPC. Untreated in blue (N=3), U18666A in gray (N=3), HP-b-CD treated in gray (N=3), U18666A and HP-b-CD treated in red (N=3). Blue * p<0.05 vs. Ctrl, Black * p<0.05 vs. U18666A. B) Venn diagram of the protein overexpressed in cells treated with U18666A, HP-b-CD and combined U18666A plus HP-b-CD. C) Venn diagram of the proteins decreased in cells treated with U18666A, HP-b-CD and U18666A plus HP-b-CD. D) Distribution of the pathways that are decreased or increased by the treatment. (Complete list provided in Table S2).

From the mass spectrometry data of the combined U18666A & HP-b-CD treatment of cells, a total of 239 proteins were significantly altered compared to control, where 171 increased and 68 decreased (Table S2). The comparison of differentially elevated (Figure 4B) and decreased (Figure 4C) proteins suggest a strong overlap between HP-b-CD induced changes and the combined U18666A & HP-b-CD treated cell proteomes. In this comparison 123 of the elevated proteins and 51 of the decreased proteins are shared between HP-b-CD and U18666A & HP-b-CD groups (Figure 4B–C).

When performing pathway analysis on the significantly increased and decreased proteins in U18666A & HP-b-CD treated cells compared to control, most pathways belong to the broad category of metabolism (Figure 4D, 43% of the increased and 46% of the reduced proteins). Beyond the similarity among the proteins modified in HP-b-CD and U18666A & HP-b-CD only groups, hypoxia and exocytosis induced by HP-b-CD are also enriched in U18666A & HP-b-CD. Their similar representation, (2–3% and 5–6% of increased pathways, Figure 3D, 4D), suggest their induction is not affected by U18666A treatment advocating for a NPC1 independent mechanism. Unlike in the HP-b-CD only treated group, sterol/lipid metabolism related pathways were identified, and accounted for 8% of the increased pathways. This proportion is in stark contrast compared to the 60% identified in U18666A (Figure 2). This major decrease in the lipid/sterol machinery in U18666A & HP-b-CD compared to U18666A group supports the known and newly identified mechanism action of HP-b-CD leading to the restoration of cholesterol homeostasis. First, HP-b-CD may induce lysosomal secretion,9 supported by the increase in exocytosis related proteins (U18666A = 0% to U18666A & HP-b-CD = 5%) and decrease in lysosome pathways (U18666A = 18% to U18666A & HP-b-CD = 5%) among the significantly increased pathways (Figure 2B, 4D, Table S2). Secondly, HP-b-CD induced NPC1 accumulation, the target of U18666A, potentially dilute the inhibitor activity therefore increasing cholesterol efflux out of the lysosome toward a homeostatic biodistribution in the cell.11

New to this study are several altered proteins associated with HP-b-CD treatment in the context of NPC disease. Combined U18666A & HP-b-CD treatment revealed 26 proteins that were initially elevated then normalized (ACAT2, AKT3, AVL9, CHCHD2, CSNK2B, CUL4B, CYP51A1, DPP6, ECH1, FSCN1, HIST2H3D, LANCL1, LMAN2, LRRC57, LYPLA2, MVK, NDUFA4, OAT, PCMT1, PGM1, PSMB7, RAB7A, RNASEH2A, RPL36A, UBE3C, WDR61). Whereas 13 proteins decreased in U18666A treatment (CDH2, DDX21, DHX15, HDDC2, HPS6, LMNA, MRPS36, NDUFAF2, PLXNB2, SCG2, SCG3, TAGLN, UAP1) were no longer significantly modified when HP-b-CD was applied. Three of the proteins significantly overexpressed in the U18666A treated cells and decreased toward normalization by HP-b-CD: ACAT2 (acetyl-CoA acetyltransferase 2), MVK (mevalonate kinase) and CYP51A1 (lanosterol 14-alpha demethylase) belong to the cholesterol synthesis machinery. These measurements suggest HP-b-CD may interfere with the early steps of the cholesterol synthesis while previous studies validated changes at the protein levels only in the later (DHCR24).24

Ten of the proteins significantly increased in the U18666A treated cells and decreased toward normalization by HP-b-CD were ACAT2, CUL4B (Cullin-4B), ECH1 (Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase), FSCN1 (Fascin), LMAN2 (Vesicular integral-membrane protein VIP36), LRRC57 (Leucine-rich repeat-containing protein 57), LYPLA2 (cyl-protein thioesterase 2), PCMT1 (Protein-L-isoaspartate(D-aspartate) O-methyltransferase), PGM1 (Phosphoglucomutase-1) and RAB7A (Ras-related protein Rab-7a), all of which associate to extracellular vesicles. These findings suggest that in NPC neuron like cells, extracellular vesicles are trapped, participating the vesicular overload characteristic of the disease. The decrease of these proteins following HP-b-CD treatment supports the increased exocytosis pathways and reduced storage potential by alleviating the release of the trapped exosomes.

Discussion

In the present study, we applied an in vitro model widely used in neurobiology to gain a better insight into the mechanism(s) of action of HP-b-CD, a cyclic oligosaccharide that has shown promise for NPC disease and is currently in clinical trials using mass spectrometry-based proteomic strategies. Additionally, present new proteomic data of differentiated and undifferentiated SH-SY5Y cells that might prove useful in evaluating other neurodegenerative disease models and potential therapeutic treatments. Overall, our results agree with the existing literature,14,20,21 and provide additional data and insight into this neuronal-like model as it is used broadly in different fields of biology.

The results obtained in the U18666A treated cells are mostly in accordance with the literature. The NPCD phenotype is extensively known to lead to the induction of the cholesterol synthesis machinery,30 lysosome accumulation and innate immune response.31,32 These mechanisms are directly related to the lysosomal sequestration of unesterified cholesterol. This mis-location of cholesterol is due to the lack of efflux out of the lysosome and is sensed by the cell as a deficiency30 and leads to the activation of innate immune sensors such as Toll-like receptors and STING.31,32 While these results were expected, NPC phenotype in the same cell line was previously reported to be associated with increased cell death15 not observed here. This discrepancy might come the method used to evaluate cell phenotype or the samples processing. Indeed, in our experiment the dead cells may have been removed during the extensive sample preparation.

The proteomic analysis of differentiated SH-SY5Y cells treated with HP-b-CD shows an increase in proteins known to be elevated in other NPC models. This observation, if confirmed in additional models or sample types, might be important in the choice of the biomarkers used to follow disease progression or therapeutic intervention. Among the selected list of protein increased in NPC are LAMP2 and SCARB2. Despite the restoration of endo/lysosomal cholesterol with HP-b-CD treatment, these proteins remain elevated, albeit closer to control levels. It is important to note that in this study, we are using an in vitro based model with one cell line, reducing the variability of the measurement. It is expected that measurement of the same proteins in patients will be significantly more heterogenous due to individual and disease severity variability in more complex systems. Therefore, it is crucial to evaluate the effect of a treatment on control models unaffected by NPC to validate that potential biomarkers used to follow the disease are not affected by the treatment.

Interestingly, in the context of NPC disease, NPC1 and NPC2 proteins are overexpressed following HP-b-CD treatment (Table S2). The induction of these proteins may participate to the mechanism of action of the drug in laboratory models and patients expressing mutated version of the proteins, with the NPC1-I1061T point mutation being the most common.33 Inducing the expression of these proteins is the rationale behind the evaluation of HDAC inhibitor (HDACi) such as valproic acid and vorinostat.29 The overlapping mechanism of action between these two approaches, HP-b-CD and HDACi, may partially explain that beneficial activity of the treatment is carried by combination therapy.34 In addition, we also observed that between 5% and 6% of the pathways induced by HP-b-CD in control or NPC cells relate to exocytosis. Inducing exocytosis has previously been shown to reduce the unesterified cholesterol load in in vitro models of NPC.35 Therefore, this mechanism may participate to the cholesterol lowering activity of HP-b-CD and further explain how this compound reduces the intracellular storage. In the present work, the proteomic data suggest that extracellular vesicles may be trapped within the cell following the loss of function of NPC. Therefore, promoting exocytosis, through an NPC1 independent mechanism may participate in the reduction of the intracellular lipid storage. Altogether the present work demonstrated the relevance of the model and of proteomic analysis to investigate drug mechanism of action in an untargeted and unbiassed manner.

Supplementary Material

Supplemental Information

Figure S1: Experimental workflow diagram

Table S2
Table S1

Acknowledgements

The authors acknowledge funding for this study from the National Institutes of Health (R01NS124784 NINDS and R01NS114413 NINDS/NIA), the Ara Parseghian Medical Research Fund at Notre Dame, and Supporting of Accelerated Research for NPC. MRP acknowledges support from the UIC Diversifying Higher Education Faculty in Illinois Fellowship.

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Associated Data

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Supplementary Materials

Supplemental Information

Figure S1: Experimental workflow diagram

NIHMS2080134-supplement-Supplemental_Information.docx (170.5KB, docx)
Table S2
NIHMS2080134-supplement-Table_S2.xlsx (1.8MB, xlsx)
Table S1
NIHMS2080134-supplement-Table_S1.xlsx (1.9MB, xlsx)

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