Accumulation of alkyl-lysophosphatidylcholines in Niemann-Pick disease type C1

Abstract

Lysosomal function is impaired in Niemann-Pick disease type C1 (NPC1), a rare and inherited neurodegenerative disorder, resulting in late endosomal/lysosomal accumulation of unesterified cholesterol. The precise pathogenic mechanism of NPC1 remains incompletely understood. In this study, we employed metabolomics to uncover secondary accumulated substances in NPC1. Our findings unveiled a substantial elevation in the levels of three alkyl-lysophosphatidylcholine [alkyl-LPC, also known as lyso-platelet activating factor (PAF)] species in NPC1 compared to controls across various tissues, including brain tissue from individuals with NPC1, liver, spleen, cerebrum, cerebellum, and brain stem from NPC1 mice, as well as in both brain and liver tissue from NPC1 cats. The three elevated alkyl-LPC species were as follows: LPC O-16:0, LPC O-18:1, and LPC O-18:0. However, the levels of PAF 16:0, PAF 18:1, and PAF 18:0 were not altered in NPC1. In the NPC1 feline model, the brain and liver alkyl-LPC levels were reduced following 2-hydroxypropyl-β-cyclodextrin (HPβCD) treatment, suggesting that alkyl-LPCs are secondary storage metabolites in NPC1 disease. Unexpectedly, cerebrospinal fluid (CSF) levels of LPC O-16:0 and LPC O-18:1 were decreased in individuals with NPC1 compared to age-appropriate comparison samples, and their levels were increased in 80% of participants 2 years after intrathecal HPβCD treatment. The fold increases in CSF LPC O-16:0 and LPC O-18:1 levels were more pronounced in responders compared to nonresponders. This study identified alkyl-LPC species as secondary storage metabolites in NPC1 and indicates that LPC O-16:0 and LPC O-18:1, in particular, could serve as potential biomarkers for tracking treatment response in NPC1 patients.

Keywords: Niemann-Pick disease type C; alkyl-lysophosphatidylcholine; biomarker; mass spectrometry; structural identification.

Fig. 1

Strategy of biomarker discovery with untargeted metabolomics. HILIC, hydrophilic interaction liquid chromatography; RPLC, reversed phase liquid chromatography.

Fig. 2

Comparison of endogenous and synthetic alkyl-LPC. The LC-HRMS2 of [M+H]⁺ ions of endogenous LPC O-16:0 (A), endogenous LPC O-18:1 (B), and endogenous LPC O-18:0 (C), synthetic LPC O-16:0 (D), synthetic LPC O-18:1 (E), and synthetic LPC O-18:0 (F). Proposed structures of product ions are given.

Fig. 3

Alkyl-LPCs in human brain measured in the biomarker verification. LPC O-16:0, LPC O-18:1, and LPC O-18:0 in dorsolateral prefrontal cortex (red), hippocampus (blue), cerebellum (green) from NPC1 patients (square) and control subjects (circle). The data are presented as mean ± standard deviation. Comparison of NPC1 and control was performed with t test with Welch’s correction. P-values < 0.05 are given above brackets.

Fig. 4

Alkyl-LPCs in mouse tissues. LPC O-16:0 (A), LPC O-18:1 (B), and LPC O-18:0 (C) in plasma, liver, spleen, cerebrum, cerebellum, and brain stem collected from NPC1 mice (n = 9) and wild-type mice (n = 6). Three NPC1 plasma samples were excluded from the analysis due to severe hemolysis of the blood. Data are presented as mean ± standard error of the mean. Data comparison for alkyl-LPCs was performed with t test. P-values < 0.05 are given above brackets.

Fig. 5

Alkyl-LPCs in cat brain and liver. (A) LPC O-16:0, LPC O-18:1, and LPC O-18:0 in brains of untreated NPC1 cats (n = 6), NPC1 cats treated with IC 120 mg HPβCD (n = 3), NPC1 cats treated with IC 120 mg HPβCD and SC 1000 mg/kg HPβCD (n = 7), and normal cats (n = 10). Data are presented as mean ± standard error of the mean. Data comparison for LPC O-16:0 and LPC O-18:0 was performed with Brown-Forsythe and Welch ANOVA test with Dunnett's T3 multiple comparisons test as post hoc test. Data comparison for LPC O-18:1 was performed with Kruskal-Wallis test with Dunn’s multiple comparisons test as post hoc test. P-values < 0.05 are given above brackets. (B) LPC O-16:0, LPC O-18:1, and LPC O-18:0 in livers of untreated NPC1 cats (n = 5), NPC1 cats treated with IC 120 mg HPβCD (n = 3), NPC1 cats treated with IC 120 mg HPβCD and SC 1000 mg/kg HPβCD (n = 6), and normal cats (n = 9). Data are normalized to mean of untreated NPC1 cats and presented as mean ± standard error of the mean. Data comparison for LPC O-16:0, LPC O-18:1, and LPC O-18:0 was performed with Brown-Forsythe and Welch ANOVA test with Dunnett's T3 multiple comparisons test as post hoc test. P-values < 0.05 are given above brackets.

Fig. 6

Alkyl-LPCs in human plasma and CSF. LPC O-16:0 (A), LPC O-18:1 (B), and LPC O-18:0 (C) in plasma from NPC1 patients (n = 7) and control subjects (n = 7). Data are presented as mean ± standard deviation. t test with Welch’s correction was used to compare NPC1 and controls. LPC O-16:0 (D), LPC O-18:1 (E), and LPC O-18:0 (F) in CSF from NPC1 patients (n = 5) and control subjects (n = 10). Data are presented as median ± interquartile. Data comparison was performed with Kruskal-Wallis test with Dunn’s multiple comparisons test as post hoc test. P-values < 0.05 are given above brackets. Fold change of LPC O-16:0 (G), LPC O-18:1 (H), and LPC O-18:0 (I) at year 2 compared to visit 2. Data are presented as median ± interquartile. Statistical analysis was not performed for the fold change of LPC O-16:0 and LPC O-18:1 due to small sample size.