A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

Arun Kumar Tharkeshwar,Danielle Te Vruchte,Jesse Trekker,Jean-Paul Decuypere,Pieter Baatsen,Jarne Pauwels,Wendy Vermeire,Huiqi Lu,Francis Impens,Frances M Platt,Liesbet Lagae,David A Priestman,Peter Vangheluwe,Katlijn Vints,Johannes V Swinnen,Ragna Sannerud,Wim Annaert,Shaun Martin,Kris Gevaert

doi:10.1038/srep41408

Abstract

Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

Highlights

We found that aminolipid-coated superparamagnetic iron oxide nanoparticles (SPIONs) remained adhered to the plasma membrane (PM), whereas dimercaptosuccinic acid (DMSA)-coated SPIONs were efficiently targeted to late endosomes(LE)/LYS
Subsequent zetapotential measurements demonstrated that amino end group SPIONs are cationic between pH 2–8 and anionic between pH 9–10, with an isoelectric point of 9 while methoxy, carboxy- and DMSA- SPIONs were anionic throughout all pH ranges (Fig. 1C)
We demonstrated that thermal decomposition combined with different surface functionalizations can be successfully used to synthesize SPIONs for subsequent applications in isolating distinct subcellular compartments

Summary

Introduction

Previous attempts using nanoparticles such as colloidal iron coated with high-molecular weight dextran to magnetically isolate lysosomes (LYS) from amoeba[35] and mammalian cells[36,37] led to lower yields due to the unstable nature of nanoparticles and their increased cellular toxicity This might have been related to the synthesis method based on co-precipitation of iron salts in aqueous alkaline solutions in the presence of stabilizers[38,39,40], which fails to properly control average particle size, size distribution, surface functionalization[41], thereby increasing cellular toxicity. Omics analysis of magnetically isolated PMs and LE/LYS allows for the first time the spatial identification of quantitative and qualitative alterations in their biomolecular composition at much higher resolution and sensitivity than was previously achieved This method provides a powerful toolbox in the context of disease analysis, including lysosomal storage diseases, but potentially extended to other neurodegenerative diseases involving endo/lysosomal transport defects

Methods

Results

Conclusion