Characterizing Proteomic and Lipidomic Changes in a Mouse Model of Niemann-Pick, Type C

Niemann-Pick, type C (NPC) is a rare and fatal, autosomal recessive disorder caused by mutations in the NPC1 gene. The main biochemical hallmark of NPC1 is the accumulation of unesterified cholesterol and sphingolipids in the late endosomal/lysosomal system. Consequently, both visceral and neurological defects occur which may manifest clinically as hepatosplenomegaly, liver dysfunction and cerebellar neurodegeneration While significant progress has been made to better understand NPC1, the downstream effects of cholesterol storage and the major mechanisms that drive these pathologies remains less understood. Surprisingly, studies geared at exploring lipid alterations in NPC1 have been largely targeted, employing either thin layer chromatography or more recently triple quadrupole MS-based assays. Moreover, studies focused on the altered proteome have been largely focused on Western blot analysis, with limited mass spectrometry-based proteomic studies being performed. Within, we aimed to define the altered proteome and lipidome in NPC1 at a late stage of disease in both peripheral and central nervous system tissues using MS-based approaches. Our proteomic analyses illuminated new protein biomarkers of NPC. Focusing on the cerebellum and cerebral cortex we observe alterations in calcium biding proteins and lysosome specific proteins including PCP-2 and LIMP-2, respectively. We also observed fatty acid regulatory and synthesis proteins that were altered including: FABP5, FADS2 and FAS. Complementary work in the liver and spleen revealed new markers including LIMP-2 and RAB7A, as well as fatty acid regulatory proteins including FABP5, FABP7, FADS1, FAS, ELOV2 and ELOV5. Based on upstream regulator analysis, we hypothesized that miRNA-155 may be a regulator of many of the altered proteins observed in the liver and spleen of NPC1 mice. In orthogonal studies, we evaluated the expression of miRNA-155 and found it to be significantly altered in both the spleen and liver tissue of the NPC1 mouse model and proposed that this specific molecule may be a unique indicator of peripheral disease. It is important to note that these LC-MS proteomic analyses were done via a standard flow label-free quantitative approach demonstrating robustness and high proteome coverage. Alongside the proteomic investigation, untargeted lipidomics was performed to reveal differences beyond cholesterol and sphingolipids. Differential analysis of the liver of the NPC1 mouse model revealed numerous alterations in TG, PC, SM and FA species, among others. Leveraging our proteomic studies, we performed a targeted GC-MS analysis of free and incorporated fatty acids in liver tissue from the NPC1 null mouse model, with emphasis on the PUFAs. We observed decreased omega-3 and 6 fatty acids including DHA and AA. In brain tissue of the NPC1 null mouse model we observed alterations in many TG, PC, BMP, ACar, SM, SHexCer and FA species.Similar findings in the brain include increased AA and DHA. Further, we performed MALDI-MSI analysis on brain tissue slices using DBDA as a novel matrix to access the spatial distribution of abundant FA species as well as HexCer d18:1/24. In this analysis, we observed higher localization in the grey matter for free FA species and SHexCer d18:1/24:1, where mutant animals displayed an overall decrease compared to control animals. Combined, our MS-based proteomic and lipidomic studies provide new insight into altered biomolecules in NPC1 disease, and thereby lay a foundation on which we can start to discern how these alterations may relate to neurodegeneration and liver dysfunction. Given the recent reports of the promise of 2-hydroxypropyl-beta-cyclodextrin (HP-β-CD) for NPC, we sought to investigate proteome and lipidomic changes with chronic treatment. First, we evaluated changes of FA species in the NPC1 null mouse model and report that treatment was able to rescue altered levels of FAs and FAHFAs in Npc1-/- mice to comparable levels of control animals. Proteomic analysis revealed that HP-B-CD treatment alters the endocannabinoid neuronal synapse pathway. Moreover, the analysis of in vitro neuronal cultures suggests that HP-β-CD alters neuronal structure. The results of the later study provide a better understanding of how HP-β-CD affects synapse function and how it may modulate glutamate signaling via the endocannabinoid signaling pathway in neurons. Taken together, these studies provide insight into the biomolecular alterations in NPC1 and how they are modulated by a viable therapeutic option.