Cholesterol oxidation products are sensitive and specific blood-based biomarkers for Niemann-Pick C1 disease

Abstract

Niemann-Pick type C1 (NPC1) disease is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol in the endolysosomes. Previous studies implicating oxidative stress in NPC1 disease pathogenesis raised the possibility that nonenzymatic formation of cholesterol oxidation products could serve as disease biomarkers. We measured these metabolites in the plasma and tissues of the Npc1(-/-) mouse model and found several cholesterol oxidation products that were elevated in Npc1(-/-) mice, were detectable before the onset of symptoms, and were associated with disease progression. Nonenzymatically formed cholesterol oxidation products were similarly increased in the plasma of all human NPC1 subjects studied and delineated an oxysterol profile specific for NPC1 disease. This oxysterol profile also correlated with the age of disease onset and disease severity. We further show that the plasma oxysterol markers decreased in response to an established therapeutic intervention in the NPC1 feline model. These cholesterol oxidation products are robust blood-based biochemical markers for NPC1 disease that may prove transformative for diagnosis and treatment of this disorder, and as outcome measures to monitor response to therapy.

Fig. 1

Oxysterol structures. (A–C) 3β,5α,6β-cholestane-triol (3β,5α,6β-triol), 7β-hydroxycholesterol (7β-HC) and 7-ketocholesterol (7-KC) are generated through non-enzymatic cholesterol oxidation. (D–F) 7α-hydroxycholesterol (7α-HC), 4β-hydroxycholesterol (4β-HC) and 25-hydroxycholesterol (25-HC) can be generated through both non-enzymatic and enzymatic pathways. (G–H) 24(S)-hydroxycholesterol (24(S)-HC) and 27-hydroxycholesterol (27-HC) are produced exclusively through enzymatic cholesterol oxidation.

Fig. 2

Cholesterol oxidation products are elevated in the plasma of Npc1^−/− mice. (A) Weight gain of WT and Npc1^−/− mice. (B) Rotarod performance of WT and Npc1^−/− mice. Error bars for WT mice are contained within the bars. (C) Kaplan-Meier survival analysis of WT and Npc1^−/− mice. (D–H) 25-HC, 3β,5α,6β-triol, 7-KC, 4β-HC, 7α-HC and 7β-HC plasma concentrations measured weekly in pooled plasma samples (n=5–8 mice/group) obtained from WT and Npc1^−/− mice. For 8–10 week time points, *p≤0.05 for Npc1^−/− vs. WT control. In panels D–H the error bars represent the precision of replicate testing of pooled samples.

Fig. 3

Accumulation of non-enzymatic cholesterol oxidation products in Npc1^−/− mouse tissues. (A,B) Oxysterol concentrations in livers of 9-week-old WT and Npc1^−/− mice. (C) 3β,5α,6β-triol concentrations in the brain tissue of 9-week-old WT and Npc1^−/− mice. (D) 3β,5α,6β-triol concentrations in cerebellar tissue of 10 day, 4-week and 7-week-old WT and Npc1^−/− mice. (E) 24(S)-HC concentrations in the brain tissue of 9-week-old WT and Npc1^−/− mice. Error bars represent samples from independent mice as denoted in each panel. *p<0.05 and **p<0.001 for Npc1^−/− vs. WT.

Fig. 4

Elevated plasma concentrations of cholesterol oxidation products in human NPC1 subjects. (A) 3β,5α,6β-triol and (B) 7-KC concentrations in plasma samples in age-matched control (n=25), NPC1 subjects (n=25), and heterozygotes (n=25). (C) Mean plasma 7-KC and 3β,5α,6β-triol levels for control, NPC1 and heterozygote subjects. *p<0.05 for heterozygotes vs. controls; **p<0.001 for heterozygotes vs. controls, and NPC1 vs. controls. (D) 7-KC concentrations as a function of 3β,5α,6β-triol concentrations for individual control, NPC1 and heterozygote subjects. (E) 24(S)-HC concentrations in fasting plasma samples in age-matched control and NPC1 subjects, and difference between age-matched subjects. *p<0.05 for difference between controls and NPC1 subjects. (F) Stability tests of 7-KC and 3β,5α,6β-triol in plasma samples processed in the presence and absence of BHT, or after a 24-hour delay after storage at 4°C or at room temperature. *p<0.05 for BHT/delay/room temperature vs. BHT/no delay (G,H) Diurnal variation of 7-KC and 3β,5α,6β-triol in plasma of NPC1 subjects.

Fig. 5

Plasma levels of cholesterol oxidation products in human subjects with diabetes and coronary artery disease. (A) Plasma 7-KC and 3β,5α,6β-triol levels re-plotted for individual control and NPC1 subjects (heterozygote subjects have been omitted), (B) Plasma 7-KC and 3β,5α,6β-triol levels plotted for individual control and diabetic (DM) subjects (n=34), which have been matched for age, gender, smoking status and statin usage. (C) Plasma 7-KC and 3β,5α,6β-triol levels plotted for individual control subjects and subjects with angiographically-proven coronary artery disease (CAD) (n=18), which have been matched for age, gender, smoking status and statin usage.

Fig. 6

Comparison of plasma oxysterol concentrations in NPC1 disease and other lysosomal storage diseases. (A) 3β,5α,6β-triol, (B) 7-KC and (C) 24(S)-HC concentrations in fasting plasma samples from control, NPC1, infantile neuronal ceroid lipofuscinosis (INCL), GM-1 gangliosidosis (GM-1), GM-2 gangliosidosis (GM-2) and Gaucher disease (GD) subjects. For 3β,5α,6β-triol, p<0.001 for NPC1 vs. INCL, GM-1, GM-2 and GD; for 7-KC, p<0.001 for NPC1 vs. GM-1 and GM-2 and p<0.01 for NPC1 vs. INCL

Fig. 7

CSF oxysterol profile in NPC1 and control subjects. (A) 3β,5α,6β-triol, (B) 5β,6β-epoxycholesterol, (c) 7α-27-HC and (d) 7β-27-HC concentrations in CSF from control and NPC1 subjects with established disease.

Fig. 8

Correlation of plasma oxysterol concentrations with age of NPC1 disease onset and disease severity. (A) Plasma 7-KC and (B) 3β,5α,6β-triol concentrations correlated with age of disease onset in NPC1 subjects. For 7-KC: r = −0.40, p<0.05; For 3β,5α,6β-triol, r = −0.41, p<0.05. (C) Plasma 7-KC and (D) 3β,5α,6β-triol concentrations correlated with disease severity rank in NPC1 subjects. For panels C–E, the severity rank increases with clinical severity of disease. For 7-KC: r = 0.39, p<0.05; For 3β,5α,6β-triol: r = 0.39, p<0.05. (E,F) Correlation of ratio of (E) plasma 7-KC and (F) 3β,5α,6β-triol to age-corrected 24(S)-HC values with disease severity rank in NPC1 subjects. For 7-KC: r = 0.66, p<0.001; For 3β,5α,6β-triol, r = 0.60, p<0.001.

Fig. 9

Circulating oxysterol biomarkers are decreased in response to cyclodextrin therapy. (A) Serum 7-KC and (B) 3β,5α,6β-triol concentrations were measured in untreated WT (4–16 weeks) and NPC1 (16 weeks) cats, and in NPC1 cats (16–18 weeks) treated with a single subcutaneous injection of 4000 or 8000 mg/kg at 3 weeks (n=2–4/group). *p≤0.05 for cyclodextrin-treated vs. untreated animals, and **p<0.01 for untreated NPC1 vs. WT animals.