A technique capable of label-free detection, mass spectrometry imaging (MSI) is a powerful tool for spatial investigation of native biomolecules in intact specimens. However, MSI has often been precluded from single-cell applications due to the spatial resolution limit set forth by the physical and instrumental constraints of the method. By taking advantage of the reversible interaction between the analytes and a superabsorbent hydrogel, we have developed a sample preparation and imaging workflow named Gel-Assisted Mass Spectrometry Imaging (GAMSI) to overcome the spatial resolution limits of modern mass spectrometers. With GAMSI, we show that the spatial resolution of MALDI-MSI can be enhanced ~3-6-fold to the sub-micrometer level without changing the existing mass spectrometry hardware or analysis pipeline. This approach will vastly enhance the accessibility of MSI-based spatial analysis at the cellular scale.

Koralege C Pathmasiri, Melissa R. Pergande, Fernando Tobias, Rima Rebiai, Avia Rosenhouse-Dantsker, Ernesto R Bongarzone, Stephanie M. Cologna

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum–folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum–folding environment by affecting Ca ⁺⁺ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1’s response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.

Niemann-Pick type C (NPC) disease, caused by mutations in the NPC1 or NPC2 genes, leads to abnormal intracellular cholesterol accumulation in late endosomes/lysosomes. Exogenous enrichment with lysobisphosphatidic acid (LBPA), also known as bis-monoacylglycerol phosphate, either directly or via the LBPA precursor phosphatidylglycerol (PG), has been investigated as a therapeutic intervention to reduce cholesterol accumulation in NPC disease. Here, we report the effects of stereoisomer configuration and acyl chain composition of LBPA on cholesterol clearance in NPC1-deficient cells. We find that S,R, S,S, and S,R LBPA stereoisomers behaved similarly, with all 3 compounds leading to comparable reductions in filipin staining in two NPC1-deficient human fibroblast cell lines. Examination of several LBPA molecular species containing one or two monounsaturated or polyunsaturated acyl chains showed that all LBPA species containing one 18:1 chain significantly reduced cholesterol accumulation, whereas the shorter chain species di-14:0 LBPA had little effect on cholesterol clearance in NPC1-deficient cells. Since cholesterol accumulation in NPC1-deficient cells can also be cleared by PG incubation, we used nonhydrolyzable PG analogs to determine whether conversion to LBPA is required for sterol clearance, or whether PG itself is effective. The results showed that nonhydrolyzable PG species were not appreciably converted to LBPA and showed virtually no cholesterol clearance efficacy in NPC1-deficient cells, supporting the notion that LBPA is the active agent promoting late endosome/lysosome cholesterol clearance. Overall these studies are helping to define the molecular requirements for potential therapeutic use of LBPA as an option for addressing NPC disease.

Niemann–Pick Disease Type C1 (NPC1) is a fatal, neurodegenerative disorder, characterized by lysosomal lipid accumulation and dysmyelination. Previous studies have documented some lipid abnormalities in the null mouse ( Npc1 ^−/− ) focused on the whole brain and liver. However, the specific lipidomic alterations in severely affected brain regions, such as cerebellum and isolated myelin remain understudied. We present a comprehensive LC-MS-based lipidomic analysis of the cerebellum and cortex of Npc1 ^−/− mice during disease progression stages, along with the first comprehensive characterization of the myelin lipidome in NPC1 disease. Our results reveal that the cerebellum accumulates lipid species, including sphingolipids and glycerophospholipids progressively, while the cortex shows an overall decline in lipid levels, indicating region-specific lipid dysregulation. Notably, bis(monoacylglycero)phosphates and their precursors—including lysophosphatidylglycerol and hemibismonoacylglycerophosphate exhibit significant accumulation, with a preference for docosahexaenoic acid (DHA)-containing species. Despite known cholesterol storage defects in NPC1, we observed reduced free cholesterol levels in both regions, which we attribute to myelin loss. Myelin-specific lipidomics demonstrated extensive dysregulation, particularly in cortical myelin, including severe losses in sulfatides, ether-lipids, and acylcarnitine, alongside striking accumulation of hydroxy-ceramides. These findings identify novel lipid alterations in brain subregions and myelin, offering critical insight into the lipid perturbations under the loss of NPC1, and highlight lipid targets that may be crucial for therapeutic intervention and biomarker development.