Quantitative Proteomic Analysis of Niemann-Pick Disease, Type C1 Cerebellum Identifies Protein Biomarkers and Provides Pathological Insight

Abstract

Niemann-Pick disease, type C1 (NPC1) is a fatal, neurodegenerative disorder for which there is no definitive therapy. In NPC1, a pathological cascade including neuroinflammation, oxidative stress and neuronal apoptosis likely contribute to the clinical phenotype. While the genetic cause of NPC1 is known, we sought to gain a further understanding into the pathophysiology by identifying differentially expressed proteins in Npc1 mutant mouse cerebella. Using two-dimensional gel electrophoresis and mass spectrometry, 77 differentially expressed proteins were identified in Npc1 mutant mice cerebella compared to controls. These include proteins involved in glucose metabolism, detoxification/oxidative stress and Alzheimer disease-related proteins. Furthermore, members of the fatty acid binding protein family, including FABP3, FABP5 and FABP7, were found to have altered expression in the Npc1 mutant cerebellum relative to control. Translating our findings from the murine model to patients, we confirm altered expression of glutathione s-transferase α, superoxide dismutase, and FABP3 in cerebrospinal fluid of NPC1 patients relative to pediatric controls. A subset of NPC1 patients on miglustat, a glycosphingolipid synthesis inhibitor, showed significantly decreased levels of FABP3 compared to patients not on miglustat therapy. This study provides an initial report of dysregulated proteins in NPC1 which will assist with further investigation of NPC1 pathology and facilitate implementation of therapeutic trials.

Adaptations of Energy Metabolism Associated with Increased Levels of Mitochondrial Cholesterol in Niemann-Pick Type C1-deficient Cells

“…In contrast to CHO cells, the brain strongly relies on the oxidative metabolism of glucose and might be particularly sensitive to metabolic alterations caused by mitochondrial cholesterol accumulation. Moreover, decreased levels of non-phosphorylated, active PDH in Npc1 Ϫ/Ϫ murine cerebellum (7,36) and impaired mitochondrial ATP generation in the presence of pyruvate (7) indicate additional defects in pyruvate oxidation in NPC1-deficient brain.…”

“…Ϫ/Ϫ cerebellum (7,36). If PDH inactivation contributed to the higher lactate production in NPC1-depleted CHO cells, we would expect a decrease in lactate production to the same levels in all cells by treatment with dichloroacetate, which indirectly activates PDH through inhibition of PDH kinase.…”

Niemann-Pick C disease and mobilization of lysosomal cholesterol by cyclodextrin

“…Two recent studies have reported that levels of mRNAs and proteins of several enzymes of energy metabolism are altered and that lactate levels are increased in NPC1-defi cient cerebella. Moreover, pyruvate oxidation is decreased and anaerobic glycolysis is increased during progression to the symptomatic stage of NPC disease ( 65,66 ). Thus, glucose metabolism appears to be affected at early stages of NPC disease.…”

Quantitative Proteomics of Human Fibroblasts with I1061T Mutation in Niemann–Pick C1 (NPC1) Protein Provides Insights into the Disease Pathogenesis*

“…A fully automated 12-step MudPIT run was performed on each sample using a three mobile phase system consisting of buffer A (5% ACN; 0.1% FA), buffer B (80% ACN, 0.1% FA), and buffer C (500 mM ammonium acetate, 5% ACN, 0.1% FA). The first step was a 60 min reverse-phase run, whereas subsequent steps were of 120 min duration and included steps with 10,20,30,40,50,60,70,80,90, and 100% buffer C run for 4 min at the beginning of the gradient and a last step that includes salt bump of 90% buffer C with 10% buffer B for 4 min.…”